Live bacterial vaccines resistant to carbon dioxide (co2), acidic ph and/or osmolarity for viral infection prophylaxis or treatment

ABSTRACT

The present invention relates to gram-negative bacterial mutants resistant to one or more stress conditions, including, but not limited to, CO 2 , acid pH, and high osmolarity. The present invention also relates more particularly to gram-negative bacterial mutants with reduced TNF-α induction having a mutation in one or more lipid biosynthesis genes, including, but not limited to msbB, that are rendered stress-resistant by a mutation in the zwf gene. The present invention provides compositions comprising one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. In particular, the present invention relates to methods for prophylaxis or treatment of a virally induced disease in a subject comprising administering to said subject one or more stress-resistant gram-negative bacterial mutants, preferably attenuated stress-resistant gram-negative bacterial mutants. The present invention further relates to methods for prophylaxis or treatment of a virally induced disease in a subject comprising administering to said subject one or more stress-resistant gram-negative bacterial mutants as vectors for the delivery of one or more therapeutic molecules. The methods of the invention provide more efficient delivery of therapeutic molecules by stress-resistant gram-negative bacterial mutants engineered to express said therapeutic molecules.

1. FIELD OF THE INVENTION

This invention is generally in the field of live bacterial vaccines forviral infection prophylaxis or treatment.

2. BACKGROUND OF THE INVENTION

Citation or identification of any reference herein, or any section ofthis application shall not be construed as an admission that suchreference is available as prior art to the present application.

There are three types of influenza viruses Influenza A, B, and C.Influenza types A or B viruses cause epidemics of disease almost everywinter. In the United States, these winter influenza epidemics can causeillness in 10% to 20% of people and are associated with an average of36,000 deaths and 114,000 hospitalizations per year. Influenza type Cinfections cause a mild respiratory illness and are not thought to causeepidemics. Influenza type A viruses are divided into subtypes based ontwo proteins on the surface of the virus. These proteins are termedhemagglutinin (H) and neuraminidase (N). Influenza A viruses are dividedinto subtypes based on these two proteins. There are 16 differenthemagglutinin subtypes H1, H2, H3, H4, H6, H7, H8, H9 H10 H11 H12, H13,H14, H15 or H16 and 9 different neuraminidase subtypes N1 N2 N3 N4 N5 N6N7 N8 or N9, all of which have been found among influenza A viruses inwild birds. Wild birds are the primary natural reservoir for allsubtypes of influenza A viruses and are thought to be the source ofinfluenza A viruses in all other animals. The current subtypes ofinfluenza A viruses found in people are A(H1N1) and A(H3N2). Influenza Bvirus is not divided into subtypes.

In 1918, a new highly pathogenic influenza H1N1 pandemic swept theworld, killing an estimated 20 and 50 million people. The H1N1 subtypecirculated from 1918 until 1957 which then was replaced by viruses ofthe H2N2 subtype, which continued to circulate until 1968. Since 1968,H3N2 viruses have been found in the population. Because H1N1 virusesreturned in 1977, two influenza A viruses are presently co-circulating(Palese and Garcia-Sarstre J. Clin. Invest., July 2002, Volume 110,Number 1, 9-13). The pathogenicity of the initial 1918H1N1 has not beenequaled by any of the latter H1N1, H2N2 or H3N2 subtypes, althoughinfection from some subtypes can be severe and result in death. Bymolecular reconstruction, the genome of the 1918 flu including the aminoacid sequences of the H1 and N1 antigens is now known (Kaiser, Science310: 28-29, 2005; Tumpey et al., Science 310: 77-81, 2005).

In 1997, 2003, and again in 2004, antigenically-distinct avian H5N1influenza viruses emerged as pandemic threats to human beings. Duringeach of these outbreaks there was concern that the avian viruses wouldadapt to become transmissible from human to human. Furthermore,oseltamivir (Tamiflu®) was ineffective in 50% of avian influenzapatients in Thailand (Tran et al. N. Engl. J. Med 350: 1179, 2004) and anew mutation in the neuraminidase has been identified which causesresistance to oseltamivir. Sequence analysis of the neuraminidase generevealed the substitution of tyrosine for histidine at amino acidposition 274 (H274Y), associated with high-level resistance tooseltamivir in influenza (N1) viruses (Gubareva et al., Selection ofinfluenza virus mutants in experimentally infected volunteers treatedwith oseltamivir. J Infect Dis 2001; 183:523-531; de Jong et al.,Oseltamivir Resistance during Treatment of Influenza A (H5N1) Infection.N. Engl. J. Med. 353:2667-2672, 2005). Such changes may alter theantigenic nature of the protein and reduce the effectiveness of vaccinesnot matched to the new variant. Other avian influenza strains ofpotential danger include H1N1, H7N7 and H9N2.

The optimum way of dealing with a human pandemic virus would be toprovide a clinically approved well-matched vaccine (i.e., containing thehemagglutinin and/or neuraminidase antigens of the emerging humanpandemic strain), but this cannot easily be achieved on an adequatetimescale because of the time consuming method of conventional influenzavaccine production in chicken eggs.

2.1 Live Bacterial Vaccine Vectors

Live attenuated bacterial vaccine vectors offer an important alternativeto conventional chicken egg based vaccines. Growth on embryonated heneggs, followed by purification of viruses from allantoic fluid, is themethod by which influenza virus has traditionally been grown for vaccineproduction. More recently, viruses have been grown on cultured celllines, which avoids the need to prepare virus strains that are adaptedto growth on eggs and avoids contamination of the final vaccine with eggproteins. However, because some of the vaccine virus may be produced incanine tumor cells (e.g., MDCK), there is concern for contamination ofthe vaccine by cancer causing elements. Moreover, both must undergo alabor intensive and technically challenging purification process, with atotal production time of 3 to 6 months. Because of the time factors andscale-up, these vaccines are produced in large, but finite batches.Meeting a world-wide demand requires stockpiling of multiple batches.Therefore, traditionally produced vaccine produced before a pandemic,would likely be generated based upon an avian influenza virus and itsantigens more than a year earlier and therefore may not be well matchedto an emerging variant and could result in only partial protection.Bacterial vectors self replicate in simple growth media can be producedextremely rapidly by virtue of exponential growth and require minimalpurification such as a single centrifugation and resuspension in apharmaceutically acceptable excipient.

Human studies have shown that antibody titres against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titre of about 30-40 gives around 50%protection from infection by a homologous virus) (Potter & Oxford (1979)Br Med Bull 35: 69-75). Antibody responses are typically measured byenzyme linked immunosorbent assay (ELISA), immunoblotting,hemagglutination inhibition, by microneutralisation, by single radialimmunodiffusion (SRID), and/or by single radial hemolysis (SRH). Theseassay techniques are well known in the art.

Cellular responses to vaccination may also occur which participate inantiviral immunity. Cells of the immune system are commonly purifiedfrom blood, spleen or lymph nodes. Separate cell populations(lymphocytes, granulocytes and monocyte/macrophages and erythrocytes)are usually prepared by density gradient centrifugation throughFicoll-Hypaque or Percoll solutions. Separation is based on the buoyantdensity of each cell subpopulation at the given osmolality of thesolution. Monocytes and neutrophils are also purified by selectiveadherence. If known subpopulations are to be isolated, for example CD4+or CD8+ T cells, fluorescence activated cell sorting (FACS) will beemployed or magnetic beads coated with specific anti-CD4 or anti-CD8monoclonal antibody are used. The beads are mixed with peripheral bloodleukocytes and only CD4+ or CD8+ cells will bind to the beads, which arethen separated out from the non-specific cells with a magnet. Anothermethod depends on killing the undesired populations with specificantibodies and complement. In some cases, a noncytotoxic antibody orother inhibitor can block the activity of a cell subtype.Characterization of cell types and subpopulations can be performed usingmarkers such as specific enzymes, cell surface proteins detected byantibody binding, cell size or morphological identification. Purified orunseparated lymphocytes can be activated for proliferation and DNAsynthesis is measured by ³H-thymidine incorporation. Other measures ofactivation such as cytokine production, expression of activationantigens, or increase in cell size are utilized. Activation isaccomplished by incubating cells with nonspecific activators such asConcanavalin A, phytohemagglutinin (PHA), phorbol myristic acetate(PMA), an ionophore, an antibody to T cell receptors, or stimulationwith specific antigen to which the cells are sensitized.

A key activity of cellular immunity reactions to pathogens such asviruses is the development of T lymphocytes that specifically killtarget cells. These activated cells develop during in vivo exposure orby in vitro sensitization. The CTL assay consists of increasing numberof sensitized lymphocytes cultured with a fixed number of target cellsthat have been prelabeled with ⁵¹Cr. To prelabel the target cells, thecells are incubated with the radiolabel. The ⁵¹Cr is taken up andreversibly binds to cytosolic proteins. When these target cells areincubated with sensitized lymphocytes, the target cells are killed andthe ⁵¹Cr is released.

Natural killer (NK) cells are an essential defense in the early stage ofthe immune response to pathogens. NK cells are active in naïveindividuals and their numbers can be enhanced in certain circumstances.The NK assay typically uses a ⁵¹Cr-labeled target and is similar to theCTL assay described above.

Specifically activated lymphocytes synthesize and secrete a number ofdistinctive cytokines. These are quantitated by various ELISA methods.Alternatively, induced cytokines are detected by fluorescence activatedflow cytometry (FACS) using fluorescent antibodies that enterpermeabilized cells. Activated cells also express new cell surfaceantigens where the number of cells is quantitated by immunofluorescentmicroscopy, flow cytometry, or ELISA. Unique cell surface receptors thatdistinguish cell populations are detected by similar immunochemicalmethods or by the binding of their specific labeled ligand.

Salmonella bacteria have been recognized as being particularly useful aslive “host” vectors for orally administered vaccines because thesebacteria are enteric organisms that, when ingested, can infect andpersist in the gut (especially the intestines) of humans and animals.

As a variety of Salmonella bacteria are known to be highly virulent tomost hosts, e.g., causing typhoid fever or severe diarrhea in humans andother mammals, the virulence of Salmonella bacterial strains toward anindividual that is targeted to receive a vaccine composition must beattenuated. Attenuation of virulence of a bacterium is not restricted tothe elimination or inhibition of any particular mechanism and may beobtained by mutation of one or more genes in the Salmonella genome(which may include chromosomal and non-chromosomal genetic material).Thus, an “attenuating mutation” may comprise a single site mutation ormultiple mutations that may together provide a phenotype of attenuatedvirulence toward a particular host individual who is to receive a livevaccine composition for avian influenza. In recent years, a variety ofbacteria and, particularly, serovars of Salmonella enterica, have beendeveloped that are attenuated for pathogenic virulence in an individual(e.g., humans or other mammals), and thus proposed as useful fordeveloping various live bacterial vaccines (see, e.g., U.S. Pat. Nos.5,389,368; 5,468,485; 5,387,744; 5,424,065; Zhang-Barber et al.,Vaccine, 17; 2538-2545 (1999); all expressly incorporated herein byreference). In the case of strains of Salmonella, mutations at a numberof genetic loci have been shown to attenuate virulence including, butnot limited to, the genetic loci phoP, phoQ, cdt, cya, crp, poxA, rpoS,htrA, nuoG, pmi, pabA, pts, damA, purA, purB, purl, zwf, aroA, aroC,gua, cadA, rfc, rjb, rfa, ompR, msbB and combinations thereof.

Bacterial flagella are known to be antigenic and subject to antigenic orphase variation which is believed to help a small portion of thebacteria in escaping the host immune response. The bacterial flagellarantigens are referred to as the H1 and H2 antigens. To avoid confusionwith the viral hemagglutinin H antigen, the bacterial flagellar Hantigen will be referred to as fH henceforth. Because theSalmonella-based vaccination of a heterologous antigen is dependent uponthe bacteria's ability to colonize the gut, which may be reduced do tothe initial immune response, the vaccination ability of the secondimmunization may be diminished due to an immune response to the vector.In Salmonella Hin invertase belongs to the recombinase family, whichincludes Gin invertase from phage Mu, Cin invertase from phage P1, andresolvases from Tn3 and the transposon (Glasgow et al. 1989, p. 637-659.In D. E. Berg, and M. M. Howe (ed.), Mobile DNA. American Society forMicrobiology, Washington, D.C.). Hin promotes the inversion of achromosomal DNA segment of 996 by that is flanked by the 26-bp DNAsequences of hixL and hixR (Johnson and Simon. 1985. Cell 41:781-791).Hin-mediated DNA inversion in S. typhimurium leads to the alternativeexpression of the fH1 and fH2 flagellin genes known as phase variation.Hin (21 kDa) exists in solution as a homodimer and binds to hix sites asa dimer (Glasgow et al. 1989. J. Biol. Chem. 264:10072-10082). Inaddition to Hin and the two hix sites, a cis-acting DNA sequence(recombinational enhancer) and its binding protein (F is, 11 KDa) arerequired for efficient inversion in vitro (Johnson et al. 1986. Cell46:531-539). Live Salmonella vaccines have not had deletions of the hingene nor defined fH1 or fH2 antigens, nor have they been constructedsuch that they lack fH antigens altogether. Accordingly, live Salmonellavaccines have not been constructed to maximize a prime-boost strategywhich alternates or eliminates the fH antigen whereby the immuneresponse of the fH antigen of the first immunization (prime) is notspecific for the antigen of the second immunization (boost). Therefore,the boost immunization is not diminished by a rapid elimination by theimmune system, and is therefore able to persist longer and moreeffectively present the immunizing antigen.

Introduction of viral genes into bacteria results in geneticallyengineered microorganisms (GEMs) for which there may be concernregarding containment of the introduced gene in the environment and itsability to reassort. Such genes could in theory provide virulencefactors to non-pathogenic or less pathogenic viral strains if allowed torecombine under circumstances were the bacterial vaccine could co-occurat the same time in the same individual as a wild type viral infection.Thus, methods that reduce bacterial recombination and increase bacterialgenetic isolation are desirable.

Insertion sequences (IS) are genetic elements that can insert copies ofthemselves into different sites in a genome. These elements can alsomediate various chromosomal rearrangements, including inversions,deletions and fusion of circular DNA segments and alter the expressionof adjacent genes. IS200 elements are found in most Salmonella species.S. typhimurium strain LT2 has six IS200s. Salmonella typhimurium strain14028 has been described to possess an additional IS200 element atcentisome 17.7 which is absent in other commonly studied Salmonellastrains LT2 and SL1344 (Murray et al., 2004 Journal of Bacteriology,186: 8516-8523). These authors describe a spontaneous hot spot (highfrequency) deletion of the Cs 17.7 to Cs 19.9 region. Live Salmonellavaccines have not had deletions of IS200 elements which would limit suchrecombination events.

Salmonella strains are known to possess phage and prophage elements.Such phage are often capable of excision and infection of othersusceptible strains and are further capable of transferring genes fromone strain by a process known as transduction. Live Salmonella vaccineshave not had deletions in phage elements such as phage recombinaseswhich exist in Salmonella, such that the phage are no longer capable ofexcision and reinfection of other susceptible strains.

Salmonella strains are known to be capable of being infected by bacteriaphage. Such phage have the potential to carry genetic elements from oneSalmonella strain to another. Live Salmonella vaccines have notcomprised mechanisms to limit phage infection such as the implantationand constitutive expression of the P22 phage repressor C2.

Bacterial expression of the viral hemagglutinin genes was firstdescribed by Heiland and Gething (Nature 292: 581-582, 1981) and Daviset al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These authorssuggest that the recombinant protein could be used as a vaccine withoutregard to the fact that the viral genetic loci are not optimal forbacterial expression. These authors did not suggest the use of livebacterial vectors as vaccine carriers, such as the geneticallystabilized and isolated vectors of the present application, nor the useof defined flagellar antigens or no flagellar antigens. Nor did theseauthors suggest the use of secreted proteins.

Use of secreted proteins in live bacterial vectors has been demonstratedby several authors. Holland et al. (U.S. Pat. No. 5,143,830, expresslyincorporated herein by reference) have illustrated the use of fusionswith the C-terminal portion of the hemolysin A (hlyA) gene. Whenco-expressed in the presence of the hemolysin protein secretion channel(hlyBD) and a functional TolC, heterologous fusions are readily secretedfrom the bacteria. Similarly, Galen et al. (Infection and Immunity 200472: 7096-7106) have shown that a heterologous fusions to the ClyA aresecreted and immunogenic. Other heterologous protein secretion systemsinclude the use of the autotransporter family. For example, Veiga et al.(2003 Journal of Bacteriology 185: 5585-5590) demonstrated hybridproteins containing the -autotransporter domain of the immunoglogulin A(IgA) protease of Nisseria gonorrhea. Fusions to flagellar proteins havealso been shown to be immunogenic. The antigen, a peptide, usually of 15to 36 amino acids in length, is inserted into the central, hypervariableregion of the FliC gene such as that from Salmonella muenchen (Verma etal. 1995 Vaccine 13: 235-24; Wu et al., 1989 Proc. Natl. Acad. Sci. USA86: 4726-4730; Cuadro et al., 2004 Infect. Immun. 72: 2810-2816; Newtonet al., 1995, Res. Microbiol. 146: 203-216, expressly incorporated byreference in their entirety herein). Antigenic peptides are selected byvarious methods, including epitope mapping (Joys and Schodel 1991.Infect. Immune. 59: 3330-3332; Hioe et al., 1990 J. Virol. 64:6246-6251; Kaverin et al. 2002, J. Gen. Virol. 83: 2497-2505; Hulse etal. 2004, J. Virol. 78: 9954-9964; Kaverin et al. 2007, J. Virol.81:12911-12917; Cookson and Bevan 1997, J. Immunol. 158: 4310-4319,expressly incorporated by reference in their entirety herein), T-cellepitope determination (Walden, 1996, Current Opinion in Immunology 8:68-74) and computer programs such as Predict7 (Carmenes et al. 1989Biochem. Biophys. Res. Comm 159: 687-693) Pepitope (Mayrose et al.,2007. Bioinformatics 23: 3244-3246). Multihybrid FliC insertions of upto 302 amino acids have also been prepared and shown to be antigenic(Tanskanen et al. 2000, Appl. Env. Microbiol. 66: 4152-4156, expresslyincorporated by reference in its entirety herein) Modification of thefusion protein by inclusion of flanking cathepsin cleavage sites hasbeen used to facilitate release within the endosomal compartment ofantigen presenting cells (Verma et al. 1995 Vaccine 13: 235-244).Trimerization of antigens has been achieved using the T4 fibritin foldontrimerization sequence (Wei et al. 2008 J. Virology 82: 6200-6208,expressly incorporated by reference in its entirety herein).

Bacterial expression of the viral hemagglutinin genes was firstdescribed by Heiland and Gething (Nature 292: 581-582, 1981) and Daviset al., (Proc. Natl. Acad. Sci. USA 78: 5376-5380). These authors teachthat the antigens may be purified from the bacteria in order to be usedas vaccines and did not suggest the use of live attenuated bacterialvectors. Furthermore, the codon usage of the viral genome is not optimalfor bacterial expression. Accordingly, a gram-negative bacterium of theenterobacteraceae such as E. coli and Salmonella will have a differentcodon usage preference (National Library of Medicine, National Centerfor Biotechnology Information, GenBank Release 150.0 [Nov. 25, 2005])and would not be codon optimized. Further, these authors usedantibiotic-containing plasmids and did not use stable chromosomallocalization. Nor did these authors suggest heterologous fusions inorder for the bacteria to secrete the antigens.

Kahn et al. (EP No. 0863211) have suggested use of a live bacterialvaccine with in vivo induction using the E. coli nitrite reductasepromoter nirB. These authors further suggest that the antigenicdeterminant may be an antigenic sequence derived from a virus, includinginfluenza virus. However, Khan et al. did not describe a vaccine foravian influenza virus. They did not describe the appropriate antigensfor an avian influenza virus, the hemagglutinin and neuraminidase, anddid not describe how to genetically match an emerging avian influenzavirus. Furthermore, it has become apparent that certain assumptions, andexperimental designs described by Khan et al. regarding live avianinfluenza vaccines would not be genetically isolated or have improvedgenetic stability in order to provide a live vaccine for avian influenzathat would be acceptable for use in humans. For example, Khan et al.state that any of a variety of known strains of bacteria that have anattenuated virulence may be genetically engineered and employed as livebacterial carriers (bacterial vectors) that express antigen polypeptidesto elicit an immune response including attenuated strains of S.typhimurium and, for use in humans, attenuated strains of S. typhi(i.e., S. enterica serovar Typhi). In support of such broad teaching,they point to the importance of “non-reverting” mutations, especiallydeletion mutations which provide the attenuation. However, non-reversiononly refers to the particular gene mutated, and not to the genome per sewith its variety of IS200, phage and prophage elements capable of avariety of genetic recombinations and/or even transductions to otherbacterial strains. Khan et al. did not describe a bacterial strain withimproved genetic stability, nor methods to reduce genetic recombination,such as deletion of the IS200 elements. Khan et al. did not describe abacterial strain with improved genetic stability by deletion of thebacteria phage and prophage elements nor limiting their transducingcapacity. Neither did Khan et al. describe methods to minimize bacterialgenetic exchange, such as constitutive expression of the P22 C2 phagerepressor.

The above comments illustrate that Khan et al. have not provided thefield with an effective vaccine against avian influenza. Clearly, needsremain for a genetically isolated and genetically stable, orallyadministered vaccine against avian influenza which is capable of rapidgenetically matching an emerging pathogenic variant.

Bermudes (WO/2008/039408), expressly incorporated herein in itsentirety, describes live bacterial vaccines for viral infectionprophylaxis or treatment. The bacteria described are live attenuatedbacterial strains that express one or more immunogenic polypeptideantigens of a virus. The bacteria useful for the techniques describedinclude Salmonella, Bordetella, Shigella, Yersenia, Citrobacter,Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia,Plesiomonas, and Aeromonas. Bermudes describes the serovars ofSalmonella. enterica that may be used as the attenuated bacterium of thelive vaccine compositions to include, without limitation, Salmonellaenterica serovar Typhimurium (“S. typhimurium”), Salmonella montevideo,Salmonella enterica serovar Typhi (“S. typhi”), Salmonella entericaserovar Paratyphi B (“S. paratyphi 13”), Salmonella enterica serovarParatyphi C (“S. paratyphi C”), Salmonella enterica serovar Hadar (“S.hadar”), Salmonella enterica serovar Enteriditis (“S. enteriditis”),Salmonella enterica serovar Kentucky (“S. kentucky”), Salmonellaenterica serovar Infantis (“S. infantis”), Salmonella enterica serovarPullorurn (“S. pullorum”), Salmonella enterica serovar Gallinarum (“S.gallinarum”), Salmonella enterica serovar Muenchen (“S. muenchen”),Salmonella enterica serovar Anaturn (“S. anatum”), Salmonella entericaserovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S.derby”), Salmonella enterica serovar Choleraesuis var. kunzendorf (“S.cholerae kunzendorf”), and Salmonella enterica serovar minnesota (S.minnesota).

Bermudes describes attenuating mutations useful in the Salmonellabacterial strains which may include genetic locus selected from thegroup consisting of phoP, phoQ, edt, cya, crp, poxA, rpoS, htrA, nuoG,pmi, pabA, pts, damA, purA, purB, purl, zwf, purF, aroA, aroB, aroC,aroD, serC, gua, cadA, rfc, rjb, rfa, ompR, msbB and combinationsthereof.

Although Bermudes discloses the msbB gene and the zwf gene, it was notrecognized that in Salmonella, the deletion of the msbB gene conferssensitivity to carbon dioxide (CO₂) and that deletion of zwf, a memberof the pentose phosphate pathway (Fraenkel, D. G. 1996 Glycolysis, pp189-198, In Eschericia coli and Salmonella typhimurium, F. C. Neidehardt(ed), ASM Press, Washington, D.C.), compensates for that deletion andrestores resistance to carbon dioxide without loosing the low degree oflipid A pyrogenicity (TNF-α induction) conferred by the msbB mutation.Furthermore, it was also not known that the msbB⁻ Salmonella are alsosensitive to acidic pH and osmolarity, and that the zwf mutation alsoenhances resistance to acidic pH and osmolarity. Therefore, the priorart does not teach a specific combination of these two mutations inorder to obtain CO₂ resistant bacteria. Nor would one ordinarily skilledin the arts be motivated to test for CO₂ resistance in Salmonelladeleted in msbB as there is no teaching that describes the occurrence ofsensitivity or its importance. As described herein, CO₂ and acidicpH-resistant ΔmsbB⁻ bacteria have improved survival under physiologicalconditions advantageous for penetration into gut mucosal, lymphoidal anddendridic tissues at lower doses, in order to elicit an immune responseto viral diseases.

3. SUMMARY OF THE INVENTION

The present invention provides improved live attenuated bacterialstrains that express one or more immunogenic polypeptide antigens of avirus, preferably an avian influenza virus, that is effective in raisingan immune response in animals, including mammals and birds.

In particular, one aspect of the invention relates to improved liveattenuated bacterial strains which may include Salmonella vectoringavian influenza antigens that can be administered orally to anindividual to elicit an immune response to protect the individual fromavian influenza. The invention provides gram-negative bacterial mutantsresistant to one or more stress conditions, including, but not limitedto, CO₂, acid pH, and high osmolarity. In a preferred embodiment,attenuated gram-negative bacterial mutants are provided which areresistant to CO₂, acid pH, and/or high osmolarity. In a more preferredembodiment, attenuated gram-negative bacterial msbB⁻ mutants resistantto CO₂, acid pH, and high osmolarity are provided. In a more preferredembodiment, attenuated gram-negative bacterial msbB⁻ mutants resistantto CO₂, acid pH, and high osmolarity are provided by a mutation in thepentose phosphate pathway. In a specific embodiment, attenuatedgram-negative bacterial msbB⁻ mutants resistant to CO₂, acid pH, andhigh osmolarity by deletion or disruption of the zwf gene are provided.However, it should be understood that the scope of the invention islimited by the claims, and not otherwise constricted to particulargenotypes or phenotypes.

The preferred bacteria are serovars of Salmonella. The preferredSalmonella strains of the invention are specifically attenuated by atleast one first mutation at genetic locus which, alone or incombination, results in increased sensitivity to CO₂, osmolarity and/oracidic pH combined with at least one second mutation that compensatesfor the increased sensitivity to CO₂, osmolarity and acidic pH andrestores resistance to CO₂, osmolarity and acidic pH. The attenuatingmutation resulting in sensitivity to CO₂, osmolarity and acidic pH maybe those of known lipid biosynthesis genes which exhibit a degree ofsafety in animals including but not limited to msbB (also known as mlt,waaN, lpxM), firA, kdsA, kdsB, kdtA, lpxA, lpxB, lpxC, lpxD, ssc, pmrA,and htrB. The resistance-conferring gene mutation can be any member ofthe pentose phosphate pathway, including zwf, pgl, gnd, rpe, rpiA, rpiB,tktA, tktB, talA, talB, especially those genes directly related togluconate production, including zwf, gnd and pgl, or related geneproducts that provide gluconate into the pentose pathway including gntTand other transporters for gluconate including but not limited to thehomologous gntU, gntP and idnT transporters. The invention also providesstress-resistant gram-negative bacterial mutants engineered to containand/or express one or more nucleic acid molecules encoding one or moretherapeutic molecules.

In one embodiment, stress-resistant gram-negative bacterial mutants areprovided which are facultative anaerobes or facultative aerobes. Inanother embodiment, stress-resistant gram-negative bacterial mutants areprovided which are facultative anaerobes or facultative aerobes and thatcomprise one or more nucleic acid molecules encoding one or moretherapeutic molecules. Examples of facultative anaerobes or facultativeaerobes include, but are not limited to, Salmonella typhi, Salmonellacholeraesuis, or Salmonella enteritidis.

In a specific embodiment, the present invention providesstress-resistant Salmonella mutants. Examples of Salmonella sp. whichmay be used in accordance with the invention include, but are notlimited to, Salmonella typhi, Salmonella choleraesuis, or Salmonellaenteritidis. Preferably, the stress-resistant Salmonella mutants areattenuated by introducing one or more mutations in one or more genes inthe lipopolysaccharide (LPS) biosynthetic pathway, and optionally one ormore mutations to auxotrophy for one or more nutrients or metabolites.In a preferred embodiment, attenuated stress-resistant Salmonellamutants comprise a genetically modified msbB gene, express an alteredlipid A molecule compared to wild-type Salmonella sp., and induce TNF-αexpression at a level less than that induced by a wild-type Salmonellasp. The growth of attenuated stress-resistant Salmonella mutants used inaccordance with the invention may be sensitive to a chelating agent suchas, e.g., Ethylenediaminetetraacetic Acid (EDTA), EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N′,N′-Tetraacetic Acid (EGTA), orsodium citrate. For example, a chelating agent may inhibit the growth ofattenuated stress-resistant Salmonella mutants by about 25%, 50%, 80%,or 99.5% compared to the growth of a wild-type Salmonella sp.Preferably, the attenuated stress-resistant Salmonella mutants used inaccordance with the invention survive in macrophages up to about 1% ofthe level of survival of a wild-type Salmonella sp, preferably up toabout 10%, more preferably from about 10% up to about 30%, even morepreferably from about 30% up to about 50%, and most preferably up toabout 90% or even higher.

In one embodiment, the present invention provides stress-resistantSalmonella mutants comprising one or more nucleic acid moleculesencoding one or more therapeutic molecules. In a preferred embodiment,the present invention provides attenuated stress-resistant Salmonellamutants, wherein the attenuation of the stress-resistant Salmonellamutants is due, at least in part, to one or more mutations in the msbBgene. In another preferred embodiment, the present invention providesattenuated stress-resistant Salmonella mutants, wherein the attenuatedstress-resistant Salmonella mutants comprise one or more nucleic acidmolecules encoding one or more therapeutic molecules and the attenuationof the stress-resistant Salmonella mutants is due, at least in part, toone or more mutations in the msbB gene.

A therapeutic molecule may be, for example, a molecule which directlyreduces the cause of a pathological condition, one which enhances hostresponse to a condition or reduces an adverse host response due to thecondition, one which reduces the incidence of superinfection or improveshost health or immune response, or the like.

In one embodiment, the present invention provides mutant Salmonella sp.comprising a genetically modified msbB gene and a mutation characterizedby increased growth when grown under CO₂ conditions compared to themsbB⁻ mutant Salmonella designated YS1646 having ATCC Accession No.202165 (Low, et. al., 1999, Nature Biotechnology 17: 37-41; Low et al.,2004 Methods Mol. Med. 90: 47-60). In another embodiment, the presentinvention provides a mutant Salmonella sp. comprising a geneticallymodified msbB gene and a mutation characterized by increased growth whengrown in acidified media compared to the msbB⁻ mutant Salmonelladesignated YS1646 having ATCC Accession No. 202165. In yet anotherembodiment, the present invention provides mutant Salmonella sp.comprising a genetically modified msbB gene and a mutation characterizedby increased growth in media with high osmolarity compared to the msbB⁻mutant Salmonella designated YS1646 having ATCC Accession No. 202165. Inaccordance with these embodiments, the mutant Salmonella sp. may furthercomprise one or more genetically modified genes to auxotrophy and/or oneor more nucleic acid molecules encoding one or more therapeuticmolecules.

In another preferred embodiment, the present invention provides a mutantSalmonella sp. comprising a genetically modified msbB gene and agenetically modified zwf gene.

According to various embodiments, the invention provides pharmaceuticalcompositions comprising pharmaceutically acceptable carriers and one ormore stress-resistant gram-negative bacterial mutants. The inventionalso provides pharmaceutical compositions comprising pharmaceuticallyacceptable carriers and one or more stress-resistant gram-negativebacterial mutants comprising nucleotide sequences encoding one or moretherapeutic molecules. The pharmaceutical compositions of the inventionmay be used in accordance with the methods of the invention for theprophylaxis or treatment of virally induced disease. Preferably, thestress-resistant gram-negative bacterial mutants are attenuated byintroducing one or more mutations in one or more genes in thelipopolysaccharide (LPS) biosynthetic pathway, and optionally one ormore mutations to auxotrophy for one or more nutrients or metabolites.

In one embodiment, a pharmaceutical composition comprises apharmaceutically acceptable carrier and one or more attenuatedstress-resistant gram-negative bacterial mutants, wherein saidattenuated stress-resistant gram-negative bacterial mutants arefacultative anaerobes or facultative aerobes. In another embodiment, apharmaceutical composition comprises a pharmaceutically acceptablecarrier and one or more attenuated stress-resistant gram-negativebacterial mutants, wherein said attenuated stress-resistantgram-negative bacterial mutants are facultative anaerobes or facultativeaerobes and comprise one or more nucleic acid molecules encoding one ormore therapeutic molecules where the therapeutic molecule is a viralantigen.

In a specific embodiment, a pharmaceutical composition comprises apharmaceutically acceptable carrier and one or more attenuatedstress-resistant gram-negative bacterial mutants, wherein the attenuatedstress-resistant gram-negative bacterial mutants are a Salmonella sp. Inanother specific embodiment, a pharmaceutical composition comprises apharmaceutically acceptable carrier and one or more attenuatedstress-resistant gram-negative bacterial mutants, wherein the attenuatedstress-resistant gram-negative bacterial mutants are a Salmonella sp.,and the attenuated stress-resistant gram-negative bacterial mutantscomprise one or more nucleic acid molecules encoding one or moretherapeutic molecules.

In a preferred embodiment, a pharmaceutical composition comprises apharmaceutically acceptable carrier and one or more attenuatedstress-resistant Salmonella mutants. In another preferred embodiment, apharmaceutical composition comprises a pharmaceutically acceptablecarrier and one or more attenuated stress-resistant Salmonella mutants,wherein said attenuated stress-resistant Salmonella mutants comprise oneor more nucleic acid molecules encoding one or more therapeuticmolecules.

The present invention encompasses treatment protocols that provide abetter therapeutic effect than current existing vaccines. In particular,the present invention provides methods for prophylaxis or treatment ofvirally induced disease in a subject comprising administering to saidsubject and one or more stress-resistant gram-negative bacterialmutants, preferably attenuated stress-resistant gram-negative bacterialmutants. The present invention also provides methods for the prophylaxisor treatment of virally induced disease in a subject comprisingadministering to said subject one or more stress-resistant gram-negativebacterial mutants, preferably attenuated stress-resistant gram-negativebacterial mutants, wherein said stress-resistant gram-negative bacterialmutants comprise one or more nucleic acid molecules encoding one or moretherapeutic molecules.

The present invention provides methods for the enhanced delivery of oneor more therapeutic molecules in a subject comprising administering tosaid subject one or more stress-resistant gram-negative bacterialmutants, preferably attenuated stress-resistant gram-negative bacterialmutants, comprising nucleic acid molecules encoding one or moretherapeutic molecules. The methods of the present invention permit lowerdosages and/or less frequent dosing of stress-resistant gram-negativebacterial mutants (preferably attenuated stress-resistant gram-negativebacterial mutants) to be administered to a subject for prophylaxis ortreatment of virally induced disease to achieve a therapeuticallyeffective amount of one or more therapeutic molecules.

In a specific embodiment, the present invention provides a method ofprophylaxis or treatment of virally induced disease in a subject, saidmethod comprising administering to said subject an effective amount of amutant Salmonella sp. comprising a genetically modified msbB gene and amutation characterized by increased growth when grown under CO₂conditions compared to the msbB⁻ mutant Salmonella designated YS1646having ATCC Accession No. 202165. In another embodiment, the presentinvention provides a method for viral prophylaxis or treatment in asubject, said method comprising administering to said subject aneffective amount of a mutant Salmonella sp. comprising a geneticallymodified msbB gene and a mutation characterized by increased growth whengrown in acidified media compared to the msbB- mutant Salmonelladesignated YS1646 having ATCC Accession No. 202165. In accordance withthese embodiments, the mutant Salmonella sp. further comprise one ormore genetically modified genes to auxotrophy and/or one or more nucleicacid molecules encoding one or more therapeutic molecules.

In a preferred embodiment, the present invention provides a method ofprophylaxis or treatment of virally induced disease in a subject, saidmethod comprising administering to said subject an effective amount of amutant Salmonella sp. comprising a genetically modified msbB gene and agenetically modified zwf gene. In accordance with this embodiment, themutant Salmonella sp. may further comprise one or more geneticallymodified genes to auxotrophy and/or one or more nucleic acid moleculesencoding one or more therapeutic molecules.

In a preferred embodiment of the invention, the bacteria have geneticmodifications which result in the expression of at least onehemagglutinin and/or one neuraminidase, where each gene is optimized forbacterial expression in at least one codon. In a most preferredembodiment, the hemagglutinin and/or neuraminidase genes are furthermodified to be secreted by the bacteria as heterologous fusion proteins.In a most preferred embodiment, the neuraminidase and hemagglutininheterologous fusion proteins are integrated into the chromosome in deltaIS200 sites.

In a preferred embodiment, the bacterial strains are geneticallystabilized by deletion of IS200 elements, which reduces their geneticrecombination potential.

In another embodiment, the bacterial strains are genetically stabilizedby deletion of phage and prophage elements, which reduces their geneticrecombination and transduction potential.

In another embodiment, the bacterial strains are genetically isolatedfrom phage infection by constitutive expression of the P22 C2 repressor,which reduces their ability to be infected by phage and the subsequenttransduction of genes by such phage.

In another embodiment, the bacterial strains have genetically definedflagellar antigens, or no flagellar antigens, which reduces the immunesystem elimination of the vector, enhancing its immunization potentialin second immunizations.

In a preferred embodiment, the genetically modified bacteria are used inanimals, including humans, birds, dogs and pigs, for protection againstavian influenza and highly pathogenic derivatives.

In another embodiment, a kit allows for rapid construction of abacterial vaccine which is closely matched to an emerging avianinfluenza or its highly pathogenic derivative.

In another embodiment, the invention provides a bacterium capable ofhaving it's growth inhibited by gluconate and a method of controllingbacterial growth by means of administering gluconate. In a preferredembodiment, the bacterium capable of having it's growth inhibited bygluconate is deficient in both the msbB and zwf genes.

The live attenuated bacteria described by Bermudes WO/2008/039408 aredesigned to achieve a close antigenic match between the vaccine strainand the target strain. Bermudes targets viruses for vaccine strainsbased on their emerging pathogenicity, and produces an effective vaccinemore closely matched to the antigen profile of the emerging pathogen. AsBermudes requires detailed knowledge of the antigenic profile of anemerging strain, such a vaccine can be produced at the time of need inorder to reduce the risk of an unmatched vaccine and potential effectsof partial protection in a human pandemic outbreak. Thus Bermudesprovides vaccines for protecting a human patient against infection by anemerging avian influenza virus strain.

Accordingly, when orally or nasally administered to an individual, alive Salmonella bacterial vaccine, in accordance with the presentinvention, that is genetically engineered to express one or more avianinfluenza antigens as described herein and having a first attenuatingmutation that reduces TNF-α induction and confers sensitivity to CO₂,osmolarity and/or acidic pH and a second mutation that confersresistance to CO₂, osmolarity and/or acidic pH and restores theirability to grow therein without increasing TNF-α induction and haveimproved ability to establish a population (infection) in thenasopharyngeal and/or bronchial/pulmonary or gut tissues and, ifproperly modified they could provide a desirable source of immunogenicavian influenza antigen polypeptide(s) to elicit an immune response inthe mucosal tissue of the individual.

The antigen(s) can invoke an antibody and/or cellular immune responsesin the patient that are capable of neutralizing the emerging avianinfluenza vaccine strains with high efficiency, as well as emergingheterologous avian influenza vaccine strains, with moderate efficiency.Preferably, the emerging avian influenza vaccine will be within the samehemagglutinin and or neuraminidase type (i.e., H1, H5, H5 (H274Y), H7 orH9 and/or N1, N2 or N7) as are the current pathogenic avian influenzastrains.

The live vaccine compositions are suitable for oral administration to anindividual to provide protection from avian influenza. Preferably, avaccine composition comprises a suspension of a live bacterial straindescribed herein in a physiologically accepted buffer or saline solutionthat can be swallowed from the mouth of an individual. However, oraladministration of a vaccine composition to an individual may alsoinclude, without limitation, administering a suspension of a bacterialvaccine strain described herein through a nasojejunal or gastrostomytube and administration of a suppository that releases a live bacterialvaccine strain to the lower intestinal tract of an individual. Vaccinesof the invention may be formulated for delivery by other various routese.g. by intramuscular injection, subcutaneous delivery, by intranasaldelivery (e.g. WO 00/47222, U.S. Pat. No. 6,635,246), intradermaldelivery (e.g. WO02/074336, W002/067983, WO02/087494, WO02/0832149WO04/016281) by transdermal delivery, by transcutaneous delivery, bytopical routes, etc. Injection may involve a needle (including amicroneedle), or may be needle-free.

Vaccines of the invention use one or more avian antigens to protectpatients against infection by an influenza virus strain that is capableof human-to-human transmission i.e. a strain that will spreadgeometrically or exponentially within a given human population withoutnecessarily requiring physical contact. The patient may also beprotected against strains that infect and cause disease in humans, butthat are caught from birds rather than from other humans (i.e., bird tohuman transmission). The invention is particularly useful for protectingagainst infection by pandemic, emerging pandemic and future panderinghuman strains e.g. for protecting against H5 and N1 influenza subtypes.Depending on the particular season and on the nature of the antigenincluded in the vaccine, however, the invention may protect against anyhemagglutinin subtypes, including H1, H2, H3, H4, H5, H6, H7, H8, H9,H10, H11, H12, H13, H14, H15 or H16 or various neuraminidase subtypes,including N1, N2, N3, N4, N5, N6, N7, N8 or N9.

The characteristics of an influenza strain that give it the potential tocause a pandemic outbreak may include: (a) it contains a new orantigenically altered hemagglutinin compared to the hemagglutinins incurrently-circulating human strains i.e., one that has not been evidentin the human population for over a decade (e.g. H2), or has notpreviously been seen at all in the human population (e.g. H5, H6 or H9,that have generally been found only in bird populations), such that thehuman population will be immunologically naive to the strain'shemagglutinin or that is a subtype which is antigenically altered bychanges in amino acid sequence or glycosylation; (b) it is capable ofbeing transmitted horizontally in the human population; (c) is capableof being transmitted from animals (including birds, dogs, pigs) tohumans; and/or (d) it is pathogenic to humans.

As a preferred embodiment of the invention protects against a strainthat is capable of human-to-human or bird-to-human or bird-to-birdtransmission, one embodiment of the invention in accordance with thataspect will generally include at least one gene that originated in amammalian (e.g. in a human) influenza virus and one gene whichoriginated in a bird or non-human vertibrate. Vaccines in accordancewith various aspects of the invention may therefore include an antigenfrom an avian influenza virus strain. This strain is typically one thatis capable of causing highly pathogenic avian influenza (HPAI). HPAI isa well-defined condition (Alexander Avian Dis (2003) 47(3 Suppl):976-81)that is characterized by sudden onset, severe illness and rapid death ofaffected birds/flocks, with a mortality rate that can approach 100%. Lowpathogenicity (LPAI) and high pathogenicity strains are easilydistinguished e.g. van der Goot et al. (Epidemiol Infect (2003)131(2):1003-13) presented a comparative study of the transmissioncharacteristics of low and high pathogenicity H5N2 avian strains. Forthe 2004 season, examples of HPAI strains are H5N1 influenza A virusese.g. A/Viet Nam/I 196/04 strain (also known as A Vietnam/3028/2004 orA/Vietnam/3028/04). The skilled person will thus be able to identify orpredict future HPAI strains and the DNA sequence and amino acidcompositions of the H and N antigens as and when they emerge. The avianinfluenza strain may be of any suitable hemagglutinin subtype, includingH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.The avian influenza strain may further be of any suitable neuraminidasesubtype N1, N2, N3, N4, N5, N6, N7, N8, or N9. The vaccines of theinvention may comprise two or more (i.e., two, three, four, or five)avian influenza hemagglutinin and neuraminidase antigens. Such avianinfluenza strains may comprise the same or different hemagglutininsubtypes and the same or different neuraminidase subtypes.

A preferred vaccine composition will contain a sufficient amount of livebacteria expressing the antigen(s) to produce an immunological responsein the patient. Accordingly, the attenuated stress-resistant Salmonellastrains described herein are both safe and useful as live bacterialvaccines that can be orally administered to an individual to provideimmunity to avian influenza and, thereby, protection from avianinfluenza.

Although not wishing to be bound by any particular mechanism, aneffective mucosal immune response to avian influenza antigen(s) inhumans by oral administration of genetically engineered, attenuatedstrains of Salmonella strains as described herein may be due to theability of such mutant strains to pass through the acidic environment ofthe stomach and persist in the intestinal tract which is known tocontain high levels of CO₂ and to exhibit acidic pH (Jensen andJorgensen, Applied and Environmental Microbiology 60: 1897-1904) beforeaccessing the gut mucosa, gut lymphoidal cells and/or gut dendridiccells. Each bacterial strain useful in the invention carries anantigen-expressing plasmid or chromosomally integrated cassette thatencodes and directs expression of one or more avian influenza antigensof avian influenza virus when resident in an attenuated Salmonellastrain described herein. As noted above, avian influenza antigens thatare particularly useful in the invention include an H1, H5, H5 (H274Y),H7 or H9 antigen polypeptide (or immunogenic portion thereof), a N1, N2or N7 antigen polypeptide (or immunogenic portion thereof), and a fusionpolypeptide comprising a heterologous secretion peptide linked in-frameto the antigenic peptide.

The serovars of S. enterica that may be used as the attenuated bacteriumof the live vaccine compositions described herein include, withoutlimitation, Salmonella enterica serovar Typhimurium (“S. typhimurium”),Salmonella montevideo, Salmonella enterica serovar Typhi (“S. typhi”),Salmonella enterica serovar Paratyphi B (“S. paratyphi 13”), Salmonellaenterica serovar Paratyphi C (“S. paratyphi C”), Salmonella entericaserovar Hadar (“S. hadar”), Salmonella enterica serovar Enteriditis (“S.enteriditis”), Salmonella enterica serovar Kentucky (“S. kentucky”),Salmonella enterica serovar Infantis (“S. infantis”), Salmonellaenterica serovar Pullorurn (“S. pullorum”), Salmonella enterica serovarGallinarum (“S. gallinarum”), Salmonella enterica serovar Muenchen (“S.muenchen”), Salmonella enterica serovar Anaturn (“S. anatum”),Salmonella enterica serovar Dublin (“S. dublin”), Salmonella entericaserovar Derby (“S. derby”), Salmonella enterica serovar Choleraesuisvar. kunzendorf (“S. cholerae kunzendorf”), and Salmonella entericaserovar minnesota (“S. Minnesota”).

By way of example, live avian influenza vaccines in accordance withaspects of the invention include known strains of S. enterica serovarTyphimurium (S. typhimurium) and S. enterica serovar Typhi (S. typhi)which are further modified as provided by the invention to form suitablevaccines for the prevention and treatment of avian influenza. SuchStrains include Ty21a, CMV906, CMV908, CMV906-htr, CMV908-htr, Ty800,aroA-/serC-, holavax, M01ZH09, VNP20009.

Novel strains are also encompassed that are attenuated in virulence bymutations in a variety of metabolic and structural genes. The inventiontherefore may provide a live vaccine composition for protecting againstavian influenza comprising a live attenuated bacterium that is a serovarof Salmonella enterica comprising, an attenuating mutation in a geneticlocus of the chromosome of said bacterium that attenuates virulence ofsaid bacterium and wherein said attenuating mutation is the Suwwandeletion (Murray et al., 2004, Journal of Bacteriology 186: 8516-8523)or combinations with other known attenuating mutations. Otherattenuating mutation useful in the Salmonella bacterial strainsdescribed herein may be in a genetic locus selected from the groupconsisting of phoP, phoQ, edt, cya, crp, poxA, rpoS, htrA, nuoG, pmi,pabA, pts, damA, purA, purB, purl, purF, aroA, aroB, aroC, aroD, serC,gua, cadA, rfc, rjb, rfa, ompR, msbB and combinations thereof.

The invention may also be incorporated into a process for preparinggenetically stable bacterial vaccines for protecting a human patientagainst infection by a avian influenza virus strain, comprisinggenetically engineering the avian antigen from an avian influenza virusstrain that can cause highly pathogenic avian influenza to comprise abacterially codon optimized expression sequence within a bacterialplasmid expression vector or chromosomal localization expression vectorand further containing engineered restriction endonuclease sites suchthat the bacterially codon optimized expression gene containssubcomponents which are easily and rapidly exchangeable in order tofacilitate rapid exchange of the genetic subcomponents to achieve a wellmatched antigen to the emerging avian influenza pathogen. The plasmidand/or chromosomal expression constructs may be further modified toresult in the secretion of the viral antigens. Administration of thevaccine to the patient invokes an antibody and/or cellular immuneresponse that is capable of neutralizing said avian influenza virusstrain.

The invention may also be incorporated into methods and compositions forproducing a bacterial vector expressing one or more avian influenzaantigens where said bacterial vector has one or more deletions in IS200elements which results in enhance genetic stability. The composition andmethods comprise a bacterial strain with a deletion in the IS200elements, such that the bacteria are no longer capable of geneticrearrangement using IS200 elements. Such a deletion is generated in anyone or more IS200 element, which is then confirmed using standardgenetic techniques.

The invention may also be incorporated into methods and compositions forproducing a genetically stabilized bacterial vector expressing one ormore avian influenza antigens where said bacterial vector has one ormore deletions in bacteria phage or prophage elements which enhancedgenetic stability and prevent phage excision. The composition andmethods comprise a bacterial strain with one or more deletions inbacteria phage or prophage elements, such that the bacteria are nolonger capable of genetic rearrangement using bacteria phage or prophageelements. Such a deletion is generated in any bacteria phage or prophageelements, which is then confirmed using standard genetic techniques.Such strains have phage with reduced capacity for transduction of genesto other strains.

The invention may also be incorporated into methods and compositions forproducing a bacterial vector expressing one or more avian influenzaantigens where said bacterial vector constitutively expresses the P22phage C2 repressor, thereby preventing new infections by bacteria phageand further preventing subsequent phage transductions by these phage.

The invention may also be incorporated into live Salmonella vaccineshaving had deletions of the hin gene and/or defined fH1 or fH2 antigens,or may have been constructed such that they lack fH antigens altogether.The invention may also make use of Salmonella strains expressingnon-overlapping O-antigens, such as those of S. typhimurium (O-1, 4, 5,12) S. typhi is (Vi), or S. montevideo (O-6, 7). Changing of the outercoat may be accomplished by genetic manipulations known to those skilledin the arts. Both antigenic changes may be used together. Accordingly,the invention may also be incorporated into live Salmonella vaccinesconstructed to maximize a prime-boost strategy which alternates oreliminates the fH antigen whereby the immune response of the fH antigenof the first immunization (prime) is not specific for the anigen of thesecond immunization (boost) and likewise, the O antigen profile of thefirst immunization is not the same for the second immunization.Therefore, the boost immunization is not diminished by a rapidelimination by the immune system, and is therefore able to persistlonger and more effectively present the immunizing heterologous avianinfluenza antigen.

An embodiment of the present invention therefore may also beincorporated into methods and compositions for producing a bacterialvector expressing one or more avian influenza antigens where saidbacterial vector has a defined flagellar H antigen (fH). The compositionand methods comprise a bacterial strain with a deletion in the Hinrecombinase gene, such that the bacteria are no longer capable ofalternating between fH1 and fH2 antigens. Such a deletion is generatedin either an fH1 or fH2 serologically defined strain, which is thenreconfirmed following deletion or disruption of the hin recombinasegene. The invention may also be incorporated into methods andcompositions for producing a bacterial vector which lacks flagellarantigens generated by deletion of the fliBC genes (i.e., fH0).Therefore, an improved composition for a prime/boost strategy isprovided where the second vaccination comprises administration of avaccine where the fH antigen composition is different from the firstvaccination. In the case where the antigen is presented as a fusion withthe fliC gene, preferably only fH1 and fH2 forms are utilized; fH0 ispreferably not used.

The invention may also may also be incorporated into a method forprotecting a human patient against infection by an avian influenza virusstrain with an improved prime/boost strategy, comprising the step ofadministering to the patient a vaccine that comprises an antigen from anavian influenza virus strain that can cause highly pathogenic avianinfluenza or 1918 influenza within a bacterial vector expressing one ormore avian influenza antigens where said bacterial vector has a definedfH antigen or no fH antigen (i.e., fH1, fH2, or fH0) and/or variousnon-overlapping O-antigens. The invention may further may also beincorporated into a method of administering a second bacterial vectorexpressing one or more avian influenza antigens comprising a second stepwhere the second administration where said bacterial vector has adefined fH antigen which is different fH antigen composition than the fHantigen of the first administration or no fH antigen. The secondadministration includes a bacterial vaccine where the first vaccineadministration is a bacterial vaccine of the present invention or isanother vaccine not encompassed by the present application, e.g.,another bacterial vaccine or an egg-based vaccine.

Similarly, the invention may also may also be incorporated into a kitcomprising (a) a first container comprising a bacterial expression codonoptimized antigen from a pathogenic avian influenza virus straincontaining unique genetically engineered restriction sites containedwithin either a bacterial protein expression plasmid or a bacterialchromosomal protein expression vector and (b) a second containercomprising bacterial vector(s) with one or more (e.g., fH1, fH2 or fH0)flagellar antigen(s) and/or various non-overlapping O-antigens.Component (a) will be modifiable to genetically match an emerging avianinfluenza virus using standard in vitro molecular techniques and can becombined with component (b) to generate one or more bacterial strainswith defined flagellar antigens which constitute a live vaccine. Thevariation(s) in flagellar antigens provided by the kit provide for morethan one live vaccine strain in which a first immunization (prime) usingone strain may be followed at an appropriate time such as 2 to 4 weeksby a second immunization (boost) using a second strain with a differentfH antigen or no fH antigen. The live vaccine compositions are suitablefor oral or nasal administration to an individual to provide protectionfrom avian influenza.

Preferably, the invention may also be incorporated into a vaccinecomposition comprising a suspension of a live bacterial strain describedherein in a physiologically accepted buffer or saline solution that canbe swallowed from the mouth of an individual. However, oraladministration of a vaccine composition to an individual may alsoinclude, without limitation, administering a suspension of a bacterialvaccine strain described herein through a nasojejunal or gastrostomytube and administration of a suppository that releases a live bacterialvaccine strain to the lower intestinal tract of an individual.

4. DEFINITIONS

In order that the invention may be more fully understood, the followingterms are defined.

CO₂ conditions: As used herein, the term “CO₂ conditions” refers to CO₂levels above ambient air. In particular, the term “CO₂ conditions”refers to CO₂ levels above 0.3%, 0.4%, 0.45%, 0.5%, 0.75%, 1%, 1.5%, 2%,2.5%, 3%, 3.5%, 4%, 4.5%, 5% or higher.

High osmolarity: As used herein, the term “high osmolarity” refers toosmolarity above the growth-permissive osmolarity level of LB-O and orMSB media described in the examples of approximately 100 milliosmoles,normal physiological osmolarity found in a subject (approximately 300milliosmoles), particular, to the osmolarity found in an organ or tissueof a subject. In certain embodiments, the term “high osmolarity” refersto osmolarity above approximately 340 millosmoles, 450 millosmoles, 475millosmoles, 500 millosmoles, 525 millosmoles, 550 millosmoles, 575millosmoles, 600 millosmoles or more. In certain embodiments, the termthe osmolarity-resistant gram-negative mutants are sensitive to normalphysiological osmolarity found in a subject.

Pentose Phosphate Pathway: The pentose phosphate pathway (also calledPhosphogluconate Pathway, or Hexose Monophosphate Shunt [HMP shunt]: seeFraenkel 1996, Glycolysis, In Neidhardt (ed) Escherichia coli andSalmonella, Second Ed., ASM Press, Washington, D.C., pp. 189-198,expressly incorporated by reference herein) is a process that serves togenerate NADPH and the synthesis of pentose (5-carbon) sugars. There aretwo distinct phases in the pathway. The first is the oxidative phase, inwhich NADPH is generated, and the second is the non-oxidative synthesisof 5-carbon sugars. This pathway is an alternative to glycolysis. Whileit does involve oxidation of glucose, its primary role is anabolicrather than catabolic. The primary functions of the pathway are: 1) Togenerate reducing equivalents, in the form of NADPH, for reductivebiosynthesis reactions within cells; 2) to provide the cell withribose-5-phosphate (R5P) for the synthesis of the nucleotides andnucleic acids; and 3) to metabolize pentose sugars derived from thedigestion of nucleic acids as well as to rearrange the carbon skeletonsof carbohydrates into glycolytic/gluconeogenic intermediates.

Gene comprising the pentose phosphate pathway include zwf (glucose6-phosphate dehydrogenase, EC 1.1.1.49), pgl (6-phosphogluconolactonase,EC 3.1.1.31), gnd (6-phosphogluconate dehydrogenase, EC 1.1.1.4), rpe(ribulose phosphate 3-epimerase, EC 5.1.3.1), rpiA/rpiB(ribose-5-phosphate isomerase A & B, EC 5.3.1.6), tktA/tktB(transkeolase A & B. EC 2.2.1.1), and talA/talB (transaldolase A & B, EC2.2.1.2). Additionally, related gene products that provide gluconateinto the pentose pathway, gntT and other transporters for gluconateincluding the homologous gntU, gntP and idnT transporters areencompassed.

Stress-resistant gram-negative bacterial mutants: As used herein, the“stress-resistant gram-negative bacterial mutants” and variationsthereof refer to gram-negative bacteria with the ability to grow underone or more environmental stresses such as may exist in the body of ananimal (mammal, bird, reptile). Examples of environmental stressesinclude, but are not limited to, CO₂ concentration, temperature, pH, andosmolarity. Stress-resistant gram-negative bacterial mutants include,but are not limited to, gram-negative bacteria that are resistant to CO₂and/or acid pH.

In a preferred embodiment, stress-resistant gram-negative mutants areattenuated. In another preferred embodiment, stress-resistantgram-negative mutants have one or more mutations in lipid metabolism, inparticular, LPS biosynthesis. In a specific embodiment, stress-resistantgram-negative mutants are stress-resistant Salmonella sp. In a preferredembodiment, stress-resistant gram-negative mutants are attenuatedstress-resistant Salmonella sp.

As used herein, “attenuated”, “attenuation”, and similar terms refer toelimination or reduction of the natural virulence of a bacterium in aparticular host organism, such as a mammal. “Virulence” is the degree orability of a pathogenic microorganism to produce disease in a hostorganism. A bacterium may be virulent for one species of host organism(e.g., a mouse) and not virulent for another species of host organism(e.g., a human). Hence, broadly, an “attenuated” bacterium or strain ofbacteria is attenuated in virulence toward at least one species of hostorganism that is susceptible to infection and disease by a virulent formof the bacterium or strain of the bacterium. As used herein, the term“genetic locus” is a broad term and comprises any designated site in thegenome (the total genetic content of an organism) or in a particularnucleotide sequence of a chromosome or replicating nucleic acid molecule(e.g., a plasmid), including but not limited to a gene, nucleotidecoding sequence (for a protein or RNA), operon, regulon, promoter,regulatory site (including transcriptional terminator sites, ribosomebinding sites, transcriptional inhibitor binding sites, transcriptionalactivator binding sites), origin of replication, intercistronic region,and portions therein. A genetic locus may be identified andcharacterized by any of a variety of in vivo and/or in vitro methodsavailable in the art, including but not limited to, conjugation studies,crossover frequencies, transformation analysis, transfection analysis,restriction enzyme mapping protocols, nucleic acid hybridizationanalyses, polymerase chain reaction (PCR) protocols, nuclease protectionassays, and direct nucleic acid sequence analysis. As used herein, theterm “infection” has the meaning generally used and understood bypersons skilled in the art and includes the invasion and multiplicationof a microorganism in or on a host organism (“host”, “individual”,“patient”) with or without a manifestation of a disease (see,“virulence” above). Infectious microorganisms include pathogenicviruses, such as avian influenza, that can cause serious diseases wheninfecting an unprotected individual. An infection may occur at one ormore sites in or on an individual. An infection may be unintentional(e.g., unintended ingestion, inhalation, contamination of wounds, etc.)or intentional (e.g., administration of a live vaccine strain,experimental challenge with a pathogenic vaccine strain). In avertebrate host organism, such as a mammal, a site of infectionincludes, but is not limited to, the respiratory system, the alimentarycanal (gut), the circulatory system, the skin, the endocrine system, theneural system, and intercellular spaces. Some degree and form ofreplication or multiplication of an infective microorganism is requiredfor the microorganism to persist at a site of infection. However,replication may vary widely among infecting microorganisms. Accordingly,replication of infecting microorganisms comprises, but is not limitedto, persistent and continuous multiplication of the microorganisms andtransient or temporary maintenance of microorganisms at a specificlocation. Whereas “infection” of a host organism by a pathogenicmicroorganism is undesirable owing to the potential for causing diseasein the host, an “infection” of a host individual with a live vaccinecomprising genetically altered, attenuated Salmonella bacterial strainas described herein is desirable because of the ability of the bacterialstrain to elicit a protective immune response to antigens of avianinfluenza virus that cause avian influenza in humans and other mammals.

As used herein, the terms “disease” and “disorder” have the meaninggenerally known and understood in the art and comprise any abnormalcondition in the function or well being of a host individual. Adiagnosis of a particular disease or disorder, such as avian influenza,by a healthcare professional may be made by direct examination and/orconsideration of results of one or more diagnostic tests.

A “live vaccine composition”, “live vaccine”, “live bacterial vaccine”,and similar terms refer to a composition comprising a strain of liveSalmonella bacteria that expresses at least one antigen of avianinfluenza, e.g., the H antigen, the N antigen, or a combination thereof,such that when administered to an individual, the bacteria will elicitan immune response in the individual against the avian influenzaantigen(s) expressed in the Salmonella bacteria and, thereby, provide atleast partial protective immunity against avian influenza. Suchprotective immunity may be evidenced by any of a variety of observableor detectable conditions, including but not limited to, diminution ofone or more disease symptoms (e.g., respiratory distress, fever, pain,diarrhea, bleeding, inflammation of lymph nodes, weakness, malaise),shorter duration of illness, diminution of tissue damage, regenerationof healthy tissue, clearance of pathogenic microorganisms from theindividual, and increased sense of well being by the individual.Although highly desired, it is understood by persons skilled in the artthat no vaccine is expected to induce complete protection from a diseasein every individual that is administered the vaccine or that protectiveimmunity is expected to last throughout the lifetime of an individualwithout periodic “booster” administrations of a vaccine composition. Itis also understood that a live vaccine comprising a bacterium describedherein may be, at the discretion of a healthcare professional,administered to an individual who has not presented symptoms of avianinfluenza but is considered to be at risk of infection or is known toalready have been exposed to avian influenza virus, e.g., by proximityor contact with avian influenza patients or virally contaminated air,liquids, or surfaces.

The terms “oral”, “enteral”, “enterally”, “orally”, “non-parenteral”,“non-parenterally”, and the like, refer to administration of a compoundor composition to an individual by a route or mode along the alimentarycanal. Examples of “oral” routes of administration of a vaccinecomposition include, without limitation, swallowing liquid or solidforms of a vaccine composition from the mouth, administration of avaccine composition through a nasojejunal or gastrostomy tube,intraduodenal administration of a vaccine composition, and rectaladministration, e.g., using suppositories that release a live bacterialvaccine strain described herein to the lower intestinal tract of thealimentary canal.

The term “recombinant” is used to describe non-naturally altered ormanipulated nucleic acids, cells transformed, electroporated, ortransfected with exogenous nucleic acids, and polypeptides expressednon-naturally, e.g., through manipulation of isolated nucleic acids andtransformation of cells. The term “recombinant” specifically encompassesnucleic acid molecules that have been constructed, at least in part, invitro using genetic engineering techniques, and use of the term“recombinant” as an adjective to describe a molecule, construct, vector,cell, polypeptide, or polynucleotide specifically excludes naturallyexisting forms of such molecules, constructs, vectors, cells,polypeptides, or polynucleotides.

Cassette, or expression cassette is used to describe a nucleic acidsequence comprising (i) a nucleotide sequence encoding a promoter, (ii)a first unique restriction enzyme cleavage site located 5′ of thenucleotide sequence encoding the promoter, and (iii) a second uniquerestriction enzyme cleavage site located 3′ of the nucleotide sequenceencoding the promoter. The cassette may also contain a multiple cloningsite (MCS) and transcriptional terminator within the 5′ and 3′restriction endonuclease cleavage sites. The cassette may also containcloned genes of interest.

As used herein, the term “salmonella” (plural, “salmonellae”) and“Salmonella” refers to a bacterium that is a serovar of Salmonellaenterica. A number of serovars of S. enterica are known. Particularlypreferred salmonella bacteria useful in the invention are attenuatedstrains of Salmonella enterica serovar Typhimurium (“S. typhimurium”)and serovar Typhi (“S. typhi”) as described herein. As used herein, theterms “strain” and “isolate” are synonymous and refer to a particularisolated bacterium and its genetically identical progeny. Actualexamples of particular strains of bacteria developed or isolated byhuman effort are indicated herein by specific letter and numericaldesignations (e.g. strains Ty21a, CMV906, CMV908, CMV906-htr,CMV908-htr, Ty800, holavax, M01ZH09, VNP20009).

The definitions of other terms used herein are those understood and usedby persons skilled in the art and/or will be evident to persons skilledin the art from usage in the text. This invention provides live vaccinecompositions for protecting against avian influenza comprising liveSalmonella enterica serovars that are genetically engineered to expressone or more avian influenza antigen polypeptides, such as the H1, H5, H5(H274Y), H7 or H9 and N1, N2 and N7 antigens of avian influenza virus.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a flow chart depicting the selection scheme for isolationof transposon insertions used to isolate CO₂-resistant mutants.

FIG. 2A-C shows CO₂ resistance of wild type Salmonella ATCC 14028 (2A),CO₂ sensitivity of an msbB- strain derived from Salmonella ATCC 14028(2B), and CO₂-resistance of a zwf, msbB⁻ strain of Salmonella derivedfrom ATCC 14028.

FIG. 3 shows msbB⁻ confers growth-sensitivity in liquid media under CO₂conditions containing physiological amounts of salt and is suppressed byzwf.

FIG. 4 shows a β-galactosidase release assays confirm cell lysis ofmsbB⁻ Salmonella in LB in the presence of 5% CO₂ and that zwf confersresistance.

FIG. 5 shows that zwf suppresses growth sensitivity to acidic pH in LBbroth in both ambient air and 5% CO₂.

FIG. 6 shows a β-galactosidase release assays confirm cell lysis in LBbroth, pH 6.6 and that zwf confers resistance.

FIG. 7 shows a series of replica plate results for different strains ondifferent media showing zwf mutation suppresses both msbB-induced CO₂sensitivity and osmotic defects.

FIG. 8 shows a flow chart depicting the selection scheme for isolationof transposon insertions used to isolate acidic pH-resistant mutants.

FIG. 9 shows a flow chart depicting the selection scheme for isolationof transposon insertions used to isolate osmolarity-resistant mutants.

6. DETAILED DESCRIPTION OF THE INVENTION

The invention provides gram-negative bacterial mutants resistant to oneor more stress conditions, including, but not limited to, CO₂, acid pH,and/or high osmolarity. In one embodiment, the present inventionprovides gram-negative bacterial mutants resistant to CO₂, acid pH,and/or high osmolarity. In a more preferred embodiment, the presentinvention provides attenuated gram-negative bacterial mutants resistantto CO₂, acid pH, and/or high osmolarity. Preferably, thestress-resistant gram-negative bacterial mutants are attenuated byintroducing one or more mutations in one or more genes in thelipopolysaccharide (LPS) biosynthetic pathway that reduces the inductionof TNF-α, and optionally, one or more mutations to auxotrophy for one ormore nutrients or metabolites.

The invention also provides stress-resistant gram-negative bacterialmutants engineered to contain and/or express one or more nucleic acidmolecules encoding one or more therapeutic molecules. In a specificembodiment, the present invention provides stress-resistantgram-negative mutants engineered to contain and/or express one or morenucleic acid molecules encoding one or more therapeutic molecules. Inanother embodiment, the present invention provides attenuatedstress-resistant gram-negative mutants engineered to contain and/orexpress one or more nucleic acid molecules encoding one or moretherapeutic molecules. In yet another preferred embodiment, the presentinvention provides attenuated stress-resistant gram-negative mutantsengineered to contain and/or express one or more nucleic acid moleculesencoding one or more therapeutic molecules.

The invention also provides pharmaceutical compositions comprisingpharmaceutically acceptable carriers and one or more stress-resistantgram-negative bacterial mutants, preferably one or more stress-resistantgram-negative bacterial mutants. The invention also providespharmaceutical compositions comprising pharmaceutically acceptablecarriers and one or more stress-resistant gram-negative bacterialmutants, comprising nucleotide sequences encoding one or moretherapeutic molecules. The pharmaceutical compositions of the inventionmay be used in accordance with the methods of the invention forprophylaxis or treatment of virally-induced disease. Preferably, thestress-resistant gram-negative bacterial mutants are attenuated byintroducing one or more mutations in one or more genes in thelipopolysaccharide (LPS) biosynthetic pathway, and optionally one ormore mutations to auxotrophy for one or more nutrients or metabolites.

The present invention encompasses treatment protocols that provide abetter therapeutic effect than current existing vaccines. In particular,the present invention provides methods for prevention or treatment ofvirally-induced disease in a subject comprising administering to saidsubject and one or more stress-resistant gram-negative bacterialmutants. The present invention also provides methods for the for viralinfection prophylaxis or treatment in a subject comprising administeringto said subject one or more stress-resistant gram-negative bacterialmutants, preferably attenuated stress-resistant gram-negative bacterialmutants, wherein said stress-resistant gram-negative bacterial mutantscomprise one or more nucleic acid molecules encoding one or moretherapeutic molecules.

The present invention provides methods for the enhanced delivery of oneor more therapeutic molecules for prophylaxis and treatment ofvirally-induced disease comprising administering to said subject one ormore stress-resistant gram-negative bacterial mutants, comprisingnucleic acid molecules encoding one or more therapeutic molecules. Themethods of the present invention permit lower dosages and/or lessfrequent dosing of stress-resistant gram-negative bacterial mutants(preferably attenuated stress-resistant gram-negative bacterial mutants)to be administered to a subject for prophylaxis or treatment ofvirally-induced disease to achieve a therapeutically effective amount ofone or more therapeutic molecules.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers with one or more of the components of thepharmaceutical compositions of the invention. The kit further comprisesinstructions for use of the composition(s). In certain embodiments ofthe invention, the kit comprises a document providing instructions forthe use of the composition(s) of the invention in, e.g., written and/orelectronic form. Said instructions provide information relating to,e.g., dosage, methods of administration, and duration of treatment.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

For reasons of clarity, the detailed description is divided into thefollowing subsections: Stress-Resistant Gram-Negative Bacterial Mutants;Production of Stress-Resistant Gram-Negative Bacterial Mutants;Identification and Selection of Stress-Resistant Gram-Negative BacterialMutants; Genetic Modifications to Stress-Resistant Mutants withTransposon Insertions or Multicopy Plasmids; Therapeutic Molecules;Expression Vehicles Methods and Compositions for Delivery; Methods ofDetermining the Therapeutic Utility; and Kits.

6.1. Stress-Resistant Gram-Negative Bacterial Mutants

Any gram-negative bacterial with the ability to grow under one or moreenvironmental stresses such as those that exist in animals (i.e.,stress-resistant gram-negative bacterial mutants) may be used in thecompositions and methods of the invention. Examples of environmentalstresses include, but are not limited to, CO₂ resistant, acid pHresistant, and/or osmolarity resistant gram-negative bacterial mutants(methods for identifying, isolating, and producing such gram-negativebacterial are described infra). In a specific embodiment, thegram-negative bacteria used in the compositions and methods of theinvention are CO₂ resistant and/or acid pH resistant gram-negativebacterial mutants.

In a preferred embodiment, the stress-resistant gram-negative bacterialmutants used in the compositions and methods of the invention areattenuated. Any technique well-known to one of skill in the art may beused to attenuate the stress-resistant gram-negative bacterial mutants.Preferably, the stress-resistant gram-negative bacterial mutants used inthe compositions and methods of the invention are attenuated by theintroduction of one or more mutations in one or more genes in thelipopolysaccharide (LPS) biosynthetic pathway, and optionally, one ormore mutations to auxotrophy for one or more nutrients or metabolites.Examples of genes in the LPS biosynthetic pathway which may beattenuated include, but are not limited to, htrB, msbB, kdsA, kdsB,kdtA, lpxB, lpxC, and lpxD. Mutations to auxotrophy can be produced bythe introduction of one or more mutations in a gene in a biosyntheticpathway such as the leucine, isoleucine, valine, phenylalanine,tryptophan, tyrosine, arginine, uracil, or purine biosynthetic pathway.In particular, a mutation in the AroA gene can result in auxotrophy. Theattenuated stress-resistant gram-negative bacterial mutants induce lowerlevels of tumor necrosis factor-α (TNF-α) than their wild-typecounterpart (i.e., about 5% to about 40%, about 5% to about 35%, about5% to about 25%, about 5% to about 15%, or about 5% to about 10% ofTNF-α induced by wild-type) as measured by techniques well-known in theart (e.g., immunoassays such as ELISAs), and thus, avoid or reduce therisk of inducing septic shock in a subject administered a mutantbacterium for viral infection prophylaxis or treatment to said subjectin accordance with the methods of the invention.

In a preferred embodiment, the stress-resistant gram-negative bacterialmutants used in the compositions and methods of the invention induce animmune response to avian influenza. The present invention encompassescompositions and methods for prophylaxis and treatment ofvirally-induced disease using stress-resistant gram-negative bacterialmutant which replicates at physiological temperatures (i.e., 35° C. to44° C.) and induce an immune response in vitro or in vivo. Preferably,such bacteria inhibit or reduce viral burden in vivo. In accordance withthe invention, such a gram-negative bacterial mutant may be engineeredto contain or express one or more therapeutic molecules which have ananti-viral immunostimulatory activity in vivo.

While the teachings in sections of this application may referspecifically to Salmonella, the compositions and methods of theinvention are in no way meant to be restricted to Salmonella butencompass any other gram-negative bacterium to which the teachingsapply. Suitable bacteria which may be used in accordance with theinvention include, but are not limited to, Escherichia coli includingenteroinvasive Escherichia coli (e.g., enteroinvasive Escherichia coli),Shigella sp., and Yersinia enterocohtica. Thus, the reference toSalmonella in this application is intended to serve as an illustrationand the invention is not limited in scope to Salmonella.

The present invention encompasses the use of Salmonella with the abilityto grow under one or more environmental stresses such as those thatexist within the body of an animal (i.e., stress-resistant Salmonellamutants) in the compositions and methods of the invention. Examples ofenvironmental stresses include, but are not limited to, CO₂concentration, temperature, pH, and osmolarity. Preferably, theSalmonella used in the compositions and methods of the invention are CO₂resistant, acid pH resistant, and/or osmolarity resistant gram-negativebacterial mutants (methods for identifying, isolating, and producingsuch Salmonella are described infra). In a specific embodiment, theSalmonella used in the compositions and methods of the invention are CO₂resistant and/or acid pH resistant gram-negative bacterial mutants.

In a preferred embodiment, the stress-resistant Salmonella used in themethods and compositions of the invention are attenuated. Preferably,the attenuated stress-resistant Salmonella mutants used in the methodsand compositions of the invention have one or more mutations in one ormore genes which reduce the virulence and toxicity of Salmonella. In apreferred embodiment, the attenuated stress-resistant Salmonella used inthe compositions and methods of the invention have mutation(s) in one ormore genes in the lipopolysaccharide (LPS) biosynthetic pathway(preferably in the msbB gene) and optionally, have one or more mutationsto auxotrophy for one or more nutrients or metabolites, such as uracilbiosynthesis, purine biosynthesis, tyrosine biosynthesis, leucine,isoleucine biosynthesis, arginine biosynthesis, valine biosynthesis,tryptophan biosynthesis and arginine biosynthesis.

The growth of an attenuated stress-resistant Salmonella used inaccordance with the invention may be sensitive to a chelating agent suchas, e.g., Ethylenediaminetetraacetic Acid (EDTA), EthyleneGlycol-bis(β-aminoethyl Ether) N,N,N′,N′-Tetraacetic Acid (EGTA), orsodium citrate. For example, a chelating agent may inhibit the growth ofan attenuated Salmonella for viral infection prophylaxis or treatment byabout 90%, 95%, 99%, or 99.5% compared to the growth of a wild-typeSalmonella used in accordance with the invention survive in macrophagesat about 50% to about 30%, about 0% to about 10%, or about 10% to about1% of the level of survival of a wild-type Salmonella sp.

The present invention provides a mutant Salmonella sp. for viralinfection prophylaxis or treatment comprising a genetically modifiedmsbB gene and a mutation characterized by increased growth when grownunder CO₂ conditions compared to the msbB- mutant Salmonella designatedYS1646 having ATCC Accession No. 202165. The present invention alsoprovides a mutant Salmonella sp. for viral infection prophylaxis ortreatment comprising a genetically modified msbB gene and a mutationcharacterized by increased growth when grown in acidified media comparedto the msbB⁻ mutant Salmonella designed YS1646 having ATCC Accession No.202165. The present invention further provides a mutant Salmonella sp.for viral infection prophylaxis or treatment comprising a geneticallymodified msbB gene and a mutation characterized by increased growth inmedia with high osmolarity compared to the msbB⁻ mutant Salmonelladesigned YS1646 having ATCC Accession No. 202165. Such mutant Salmonellasp. may further comprise one or more genetically modified genes toauxotrophy. In a preferred embodiment, the present invention providesSalmonella mutants comprising a genetically modified msbB gene and agenetically modified zwf gene.

6.1.1. Characteristics of CO₂-Resistant Gram-Negative Bacterial Mutant

The primary characteristic of CO₂-resistant gram-negative bacterialmutants is the enhanced percentage of their recovery on LB agar in CO₂relative to the parental strain of bacteria from which they werederived. In one embodiment, the percent recovery of CO₂-resistantgram-negative mutants grown under CO₂ conditions is approximately 2% toapproximately 95%, approximately 2% to approximately 75%, approximately2% to approximately 50%, approximately 2% to about 40%, approximately 2%to about 30%, approximately 2% to about 25%, approximately 2% to about20% or about 2% to approximately 10% greater than the recovery of theparental strain of bacteria from which the CO₂-resistant gram-negativebacterial mutants were derived grown under the same conditions.

A secondary characteristic of CO₂-resistant gram-negative bacterialmutants with mutations in lipid biosynthesis genes that suppress TNF-αinduction is that the derived mutant retain the same low-level inductionof TNF-α. In one embodiment, the percent TNF-α induction isapproximately 2% to approximately 95%, approximately 2% to approximately75%, approximately 2% to approximately 50%, approximately 2% to about40%, approximately 2% to about 30%, approximately 2% to about 25%,approximately 2% to about 20% or about 2% to approximately 10% that ofthe wild type strain of bacteria grown under the same conditions.

As the pH tends to drop during incubation in 5% CO₂, some CO₂-resistantgram-negative mutants may have increased growth in acidified mediarelative to the parental strain of bacteria from which they werederived. Thus, CO₂-resistant clones may be tested for resistance toacidic pH (such as pH 6.7 or lower), utilizing the methods describedinfra. In one embodiment, CO₂-resistant gram-negative mutants growapproximately 2% to approximately 95%, approximately 2% to approximately75%, approximately 2% to approximately 50%, approximately 2% toapproximately 40%, approximately 2% to approximately 30%, approximately2% to approximately 25%, or approximately 2% to approximately 10% lessin acidified media than the parental strains of bacteria from theCO₂-resistant gram-negative mutants were derived.

In addition, some CO₂-resistant gram-negative mutants may be moreattenuated than the parental strains of bacteria from which they werederived.

6.1.2. Characteristics of Acid pH-Resistant Gram-Negative BacterialMutants

The primary characteristic of acid pH-resistant gram-negative bacterialmutants is their ability to grow in liquid media under acidic pHconditions (e.g., pH 6.7, ph 6.6, pH 6.5, pH 6.25, pH 6.0, pH 5.5, pH5.0, pH 4.5, pH 4.0, pH 3.5, pH 3.0, pH 2.5, pH 2.0, pH 1.5, pH 1.0 orlower) relative to the parental strain of bacteria from which they werederived. In one embodiment, the growth of the acid pH-resistantgram-negative mutants in acidified media is approximately 2% toapproximately 95%, approximately 2% to approximately 75%, approximately2% to approximately 50%, approximately 2% to approximately 40%,approximately 2% to approximately 30%, approximately 2% to approximately20% or approximately 2% to approximately 10% or higher than the growthof the parental strain of bacteria from which the acid pH-resistantgram-negative bacterial mutants were derived grown under the sameconditions. In a preferred embodiment, the growth of the acidpH-resistant gram-negative mutants in acidified media is approximately40% to 100% higher than the growth of the parental strain of bacteriafrom which the acid pH-resistant gram-negative bacterial mutants werederived grown under the same conditions.

A secondary characteristic of acidic pH-resistant gram-negativebacterial mutants with mutations in lipid biosynthesis genes thatsuppress TNF-α induction is that the derived mutant retain the samelow-level induction of TNF-α. In one embodiment, the percent TNF-αinduction is approximately 2% to approximately 95%, approximately 2% toapproximately 75%, approximately 2% to approximately 50%, approximately2% to about 40%, approximately 2% to about 30%, approximately 2% toabout 25%, approximately 2% to about 20% or about 2% to approximately10% that of the wild type strain of bacteria grown under the sameconditions.

Some acid pH-resistant gram-negative mutants under CO₂ conditions mayhave enhanced recovery relative to the parental strain of bacteria fromwhich they were derived under the same conditions. Thus, acidpH-resistant clones may be tested for enhanced recovery under CO₂conditions, utilizing the methods described herein.

Some acid pH-resistant gram-negative mutants may have increasedsensitivity to osmolarity. In addition, some acid pH-resistantgram-negative mutants may be more attenuated than the parental strainsof bacteria from which they were derived.

6.1.3. Characteristics of Osmolarity-Resistant Gram-Negative BacterialMutants

The primary characteristic of osmolarity-resistant gram-negativebacterial mutants is their ability to survive and/or grow in mediahaving high osmolarity relative to the parental strain of bacteria fromwhich they were derived. In one embodiment, the survival and/or growthof the osmolarity-resistant gram-negative bacterial mutants in mediahaving high osmolarity is approximately 2% to approximately 95%,approximately 2% to approximately 75%, approximately 2% to approximately50%, approximately 2% to approximately 40%, approximately 2% toapproximately 30%, approximately 2% to approximately 25%, approximately2% to approximately 20% or approximately 2% to approximately 10% betterthan the survival and/or growth of the parental strain of bacteria fromwhich the osmolarity-resistant gram-negative bacterial mutants werederived grown under the same conditions. In a preferred embodiment, thesurvival and/or growth of the osmolarity-resistant gram-negativebacterial mutants in media having high osmolarity is approximately 40%to 100% better than the survival and/or growth of the parental strain ofbacteria from which the osmolarity-resistant gram-negative bacterialmutants were derived grown under the same conditions.

A secondary characteristic of osmolarity-resistant gram-negativebacterial mutants with mutations in lipid biosynthesis genes thatsuppress TNF-α induction is that the derived mutant retain the samelow-level induction of TNF-α. In one embodiment, the percent TNF-αinduction is approximately 2% to approximately 95%, approximately 2% toapproximately 75%, approximately 2% to approximately 50%, approximately2% to about 40%, approximately 2% to about 30%, approximately 2% toabout 25%, approximately 2% to about 20% or about 2% to approximately10% that of the wild type strain of bacteria grown under the sameconditions.

Some osmolarity-resistant gram-negative bacterial mutants may haveincreased sensitivity to CO₂ and/or acid pH stress conditions relativeto the parental strains of bacteria from which they were derived.Further, some osmolarity-resistant gram-negative bacterial mutants mayhave increased sensitivity to CO₂ and/or acid pH stress conditionsrelative to the parental strains of bacteria from which they werederived, but the sensitivity of the osmolarity-resistant gram-negativebacterial mutants to CO₂ and/or acid pH stress conditions is compensatedfor by other genetic alterations (e.g., alterations which causeresistance to CO₂ and/or acid pH stress conditions).

In addition, some osmolarity-resistant gram-negative bacterial mutantsmay be more attenuated than the parental strains of bacteria from whichthey were derived.

6.2. Production of Stress-Resistant Gram-Negative Bacterial Mutants

Genetic alterations that confer resistance to one or more environmentalstresses to gram-negative bacteria, preferably attenuated gram-negativebacteria and more preferably attenuated gram-negative bacteria for viralinfection prophylaxis or treatment, can be produced utilizing any methodwell-known to one of skill in the art. For example, stress-resistantgram-negative bacterial mutants may be obtained by growing the bacteriaunder various selective pressures or by random mutagenesis (e.g., usinga transposon library, using a multicopy plasmid library or by exposingthe bacteria to various mutagens). Examples of growth conditionparameters which may be varied to obtain stress-resistant mutantsinclude, but are not limited to, the temperature, the type of media usedto grow the bacteria, the pH of the media, and the CO₂concentration/levels. Examples of mutagens which may be used to obtainstress-resistant mutants include, but are not limited to, ultravioletlight and nitrosoguanadine.

6.3 Identification and Selection of Stress-Resistant Gram-NegativeBacterial Mutants

Gram-negative bacteria, preferably attenuated gram-negative bacteria,and/or preferably attenuated gram-negative bacteria for viral infectionprophylaxis or treatment, with resistance to one or more environmentalstresses can be identified and selected for utilizing any methodwell-known to one of skill in the art. In general, a pool of bacteriawith genetic variations is subjected to one or more selection criteriaand the resistant clones are isolated. A pool of gram-negative bacteriawith genetic variations may be composed of spontaneous mutants, alibrary of transposon mutants or mutants transformed with a library ofcloned DNA in a multicopy plasmid. The selected techniques that a poolof gram-negative bacteria with genetic variations is subjected to variesdepending upon the particular stress-resistant mutant that one isattempting to select. Selection techniques for a particularstress-resistant gram-negative bacteria may include, e.g., plating thebacteria to LB agar plates under the stress condition, growing thebacteria in LB broth under the stress condition, and then plating thebacteria to LB agar plates. Individual colonies are then isolated fromthe agar plates and tested for growth under the particular stresscondition. Colonies with greater growth ability than the parental strainof bacteria from which they were derived are deemed to be resistant tothe particular stress condition. In a specific embodiment, individualcolonies of gram-negative bacteria with genetic variations are deemed tobe resistant to a particular stress condition if they grow 2 fold,preferably 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold,30 fold, 35 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold,75 fold or higher levels under the stress condition than the parentalstrain of bacteria from which they were derived. In another embodiment,individual colonies of gram-negative bacteria with genetic variationsare deemed to have resistance to a particular stress condition if theirgrowth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater under stressconditions than the parental strain of bacteria from which they werederived.

In order to distinguish spontaneous stress-resistant mutants fromtransposon and plasmid-based clones, the transposon or plasmid can betransferred to another strain using selection for the appropriateantibiotic marker found on the transposon or plasmid. Several siblingcolonies may then be isolated and tested for resistance to theparticular stress condition, thus avoiding spontaneous mutants. In orderto simultaneously transfer large numbers of transposon or plasmid-basedstress-resistant clones to distinguish them from spontaneousstress-resistant clones, following the first selection where thebacteria are grown under stress conditions, the clones may be pooledtogether, the genetic marker transferred, and then multiple siblingclones tested for growth under the stress condition.

6.3.1. Identification and Selection of CO₂-Resistant Gram-NegativeBacterial Mutants

Gram-negative bacteria, preferably attenuated gram-negative bacteria,with resistance to CO₂ can be identified and selected for utilizing anymethod well-known to one of skill in the art. In general, a pool ofbacteria with genetic variations is subjected to one or more selectioncriteria and the resistant clones are isolated. A pool of gram-negativebacteria with genetic variations may be composed of spontaneous mutants,a library of transposon mutants or mutants transformed with a library ofcloned DNA in a multicopy plasmid. Selection techniques for isolatingCO₂-resistant gram-negative bacteria may include, but are not limitedto, growing the bacteria on LB agar plates at 37° C. under CO₂conditions, growing the bacteria in LB broth at 37° C. in CO₂, and thengrowing the bacteria on LB agar plates at 37° C. under CO₂ conditions orair. Individual colonies are then isolated from the agar plates andtested for plating efficiency on LB agar at 37° C. in air and LB agar at37° C. in CO₂. Colonies with greater than 0.5%, preferably 1%, 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% recover on the LB agar at37° C. in CO₂ are deemed to have enhanced resistance to CO₂ relative tothe parental strain of bacteria from which they were derived.

In order to distinguish spontaneous CO₂-resistant mutants fromtransposon and plasmid-based clones, the transposon or plasmid can betransferred to another strain using selection for the appropriateantibiotic marker found on the transposon or plasmid. Several siblingcolonies may then be isolated and tested for resistance to CO₂, thusavoiding spontaneous mutants. In order to simultaneously transfer largenumbers of transposon or plasmid-based stress-resistant clones todistinguish them from spontaneous CO₂-resistant clones, following thefirst selection where the bacteria are grown on LB agar plates under CO₂conditions, the clones may be pooled together, the genetic markertransferred, and then multiple sibling clones tested for growth underthe CO₂ conditions.

6.3.2. Identification and Selection of Acid pH-Resistant Gram-NegativeBacterial Mutants

Gram-negative bacteria, preferably attenuated gram-negative bacteria,with resistance to acidic pH can be identified and selected forutilizing any method well-known to one of skill in the art. In general apool of bacteria with genetic variations is subjected to one or moreselection criteria and the resistant clones are isolated. A pool ofgram-negative bacteria with genetic variations may be composed ofspontaneous mutants, a library of transposon mutants or mutantstransformed with a library of cloned DNA in a multicopy plasmid.Selection techniques for isolating acid pH-resistant gram-negativebacteria may include, but are not limited to, plating the bacteria on LBagar plates at 37° C. at an acidic pH (e.g., pH 6.7, pH 6.6, pH 6.5, pH6.4, pH 6.0, pH 5.5, pH 5.0, pH 4.5, pH 4.0, pH 3.5, pH 2.0, pH 2.5 orpH 1.0), growing the bacteria in LB broth at 37° C. at an acidic pH(e.g., pH 6.7, pH 6.6, pH 6.5, pH 6.4, pH 6.0, pH 5.5, pH 5.0, pH 4.5,pH 4.0, pH 3.5, pH 2.0, pH 2.5 or pH 1.0), and then plating the bacteriaon LB agar plates at 37° C. Individual colonies are then isolated fromthe agar plates and tested for growth in acidified media at 37° C.Colonies with greater growth ability than the parental strain ofbacteria from which they were derived are deemed to be resistant to aparticular acid pH (e.g., pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH 3.5, pH2, pH 2.5 or pH 1). In a specific embodiment, individual colonies ofgram-negative bacteria with genetic variations are deemed to beresistant to an acidic pH (e.g., pH 6.5, pH 6, pH 5, pH 4.5, pH 4, pH3.5, pH 2, pH 2.5 or pH 1) if they grow 2 fold, preferably 4 fold, 6fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 45fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold, 75 fold or higherlevels in acidified media than the parental strain of bacteria fromwhich they were derived. In another embodiment, individual colonies ofgram-negative bacteria with genetic variations are deemed to haveresistance to an acidic pH if their growth is 5%, preferably 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or greater in acidified media than the parental strain ofbacteria from which they were derived.

In order to distinguish spontaneous acid pH-resistant mutants fromtransposon and plasmid-based clones, the transposon or plasmid can betransferred to another strain using selection for the appropriateantibiotic marker found on the transposon or plasmid. Several siblingcolonies may then be isolated and tested for resistance to an acidic pH,thus avoiding spontaneous mutants. In order to simultaneously transferlarge numbers of transposon or plasmid-based stress-resistant clones todistinguish them from spontaneous acid pH-resistant clones, followingthe first selection where the bacteria are grown on LB agar plates at anacidic pH, the clones may be pooled together, the genetic markertransferred, and then multiple sibling clones tested for growth inacidified media.

6.3.3. Identification and Selection of Osmolarity-ResistantGram-Negative Bacterial Mutants

Gram-negative bacteria, preferably attenuated gram-negative bacteria,and more preferably attenuated gram-negative bacteria, with resistanceto high osmolarity can be identified and selected for utilizing anymethod well-known to one of skill in the art. In general, a pool ofbacteria with genetic variations is subjected to one or more selectioncriteria and the resistant clones are isolated. A pool of gram-negativebacteria with genetic variations may be composed of spontaneous mutants,a library of transposon mutants or mutants transformed with a library ofcloned DNA in a multicopy plasmid. Selection techniques for isolatinghigh osmolarity-resistant gram-negative bacteria may include, but arenot limited to, growing the bacteria on agar plates having highosmolarity at 37° C., growing the bacteria in nutrient broth having highosmolarity at 37° C., and then growing the bacteria on agar plateshaving or not having high osmolarity at 37° C. Examples of agents thatresult in high osmolarity include, but are not limited to, salts (e.g.,NaCl or KCl) and sugars (e.g., sucrose or glucose). Individual coloniesare then isolated from the agar plates and tested for growth in mediahaving high osmolarity at 37° C. Colonies with greater growth abilitythan the parental strain of bacteria from which they were derived aredeemed to have resistance to high osmolarity. In a specific embodiment,individual colonies of gram-negative bacteria with genetic variationsare deemed to have resistance to high osmolarity if they grow to 2 fold,preferably 4 fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold,30 fold, 35 fold, 45 fold, 50 fold, 55 fold, 60 fold, 65 fold, 70 fold,75 fold or higher levels in media having high osmolarity than theparental strain of bacteria from which they were derived. In anotherembodiment, individual colonies of gram-negative bacteria with geneticvariations are deemed to have resistance to high osmolarity if theirgrowth is 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater in media havinghigh osmolarity than the parental strain of bacteria from which theywere derived.

In order to distinguish spontaneous osmolarity-resistant mutants fromtransposon and plasmid-based clones, the transposon or plasmid can betransferred to another strain using selection for the appropriateantibiotic marker found on the transposon or plasmid. Several siblingcolonies may then be isolated and tested for resistance to highosmolarity, thus avoid spontaneous mutants. In order to simultaneouslytransfer large numbers of transposon or plasmid-based stress-resistantclones to distinguish them from spontaneous osmolarity-resistant clones,following the first selection where the bacteria are grown on LB agarplates having high osmolarity, the clones may be pooled together, thegenetic marker transferred, and then multiple sibling clones tested forgrowth in media having high osmolarity.

6.4 Genetic Modifications to Stress-Resistant Mutants with TransposonInsertions

Stress-resistant gram-negative mutants with transposon insertions can bere-engineered to have a deletion and/or insertion in the same site inorder to eliminate the antibiotic resistance and transposon element.First, the site of the transposon insertion is determined using standardtechniques well-known to those skilled in the art. Such techniquesinclude, e.g., cloning from chromosomal DNA based on selection forantibiotic resistance and sequencing of the adjacent region, usingGenomeWalker™ (Clontech, Palo Alto, Calif.) or direct chromosomalsequencing (Qiagen, Valencia, Calif.). A deletion and/or insertion isthen constructed using PCR to generate the two segments necessary forthe use of the sucrase vector (Donnenberg and Kaper, 1991, Infection andImmunity 59: 4310-4317). A multiple cloning site can be engineered atthe junction of the two segments used to create an insertion. Theinsertion can be non-coding DNA or coding DNA (e.g., a nucleotidesequence encoding a therapeutic molecule such as prodrug-convertingenzyme).

The genetic modification of a spontaneous mutant may be identified usingstandard techniques well-known to one of skill in the art. One techniqueto identify the genetic modification(s) of a spontaneous mutant useslinkage to transposons, as described by Murray et al., 2001, J.Bacteriology 183: 5554-5561. Another technique to identify the geneticmodification(s) of a spontaneous mutant is to generate a DNA libraryderived from the strain of interest in a low-copy or transposon vectorand to select for resistance to a particular stress condition. Theplasmid or transposon DNA is then sequenced as described above. Anothertechnique to identify the genetic modification(s) of a spontaneousmutant is to use a Genechip approach. In the Genechip approachdifferences between the spontaneous mutant and the parental strain areidentified. The spontaneous deletion, rearrangement, duplication orother form of mutation identified in the spontaneous mutant may then bere-engineered into a multicopy plasmid such as asd vector or a sucrasechromosomal vector as described above.

6.5. Kits

Similarly, the invention may also provide a kit comprising (a) a firstcontainer comprising a bacterial expression codon optimized antigen froma pathogenic avian influenza virus strain containing unique geneticallyengineered restriction sites contained within either a bacterial proteinexpression plasmid or a bacterial chromosomal protein expression vectorand (b) a second container comprising bacterial vector(s) with one ormore (e.g., fH1, fH2 or fH0) flagellar antigen(s) and/or variousnon-overlapping O-antigens. Component (a) will be modifiable togenetically match an emerging avian influenza virus using standard invitro molecular techniques and can be combined with component (b) togenerate one or more bacterial strains with defined flagellar antigenswhich constitute a live vaccine. The variation(s) in flagellar antigensprovided by the kit provide for more than one live vaccine strain inwhich a first immunization (prime) using one strain may be followed atan appropriate time such as 2 to 4 weeks by a second immunization(boost) using a second strain with a different fH antigen or no fHantigen. The live vaccine compositions are suitable for oraladministration to an individual to provide protection from avianinfluenza.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers with one or more of the components of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In a specific embodiment of the invention, the kit comprises one or morestress-resistant gram-negative bacterial mutants and optionally means ofadministering the pharmaceutical compositions of the invention. Thedifferent stress-resistant gram-negative bacterial mutants may comprisenucleotide sequences encoding one or more therapeutic molecules. The kitmay further comprise instructions for use of said stress-resistantgram-negative bacterial mutants. In certain embodiments of theinvention, the kit comprises a document providing instruction for theuse of the composition of the invention in, e.g., written and/orelectronic form. Said instructions provide information relating to,e.g., dosage, method of administration, and duration of treatment.

In one embodiment, a kit of the invention comprises a stress-resistantgram-negative bacterial mutant in a vial and instructions foradministering the stress-resistant gram-negative bacterial mutants forviral prophylaxis or treatment, wherein the stress-resistantgram-negative bacterial mutant is a facultative anaerobe or facultativeaerobe. In accordance with this embodiment, the stress-resistantgram-negative bacterial mutant may be engineered to express one or morenucleic acid molecules encoding one or more therapeutic molecules. Inanother embodiment, a kit of the invention comprises an anti-viral agentcontained in a first vial, a stress-resistant gram-negative bacterialmutant in a second vial, and instructions for administering theanti-viral agent and stress-resistant gram-negative bacterial mutant toa subject for viral infection prophylaxis or treatment. In accordancewith this embodiment, stress-resistant gram-negative bacterial mutantmay be engineered to express one or more nucleic acid molecules encodingone or more therapeutic molecules. Preferably, the stress-resistantgram-negative bacterial mutants included in the kits of the inventionare stress-resistant gram-negative Salmonella mutants.

In another embodiment, a kit of the invention comprises an attenuatedstress-resistant gram-negative bacterial mutant in a vial andinstructions for administering the attenuated stress-resistantgram-negative bacterial mutant to a subject for viral infectionprophylaxis or treatment, wherein the attenuated stress-resistantgram-negative bacterial mutant is a facultative anaerobe or facultativeaerobe. In accordance with this embodiment, the attenuatedstress-resistant gram-negative bacterial mutant may be engineered toexpress one or more nucleic acid molecules encoding one or moretherapeutic molecules. In another embodiment, a kit of the inventioncomprises an anti-viral agent contained in a first vial, an attenuatedstress-resistant gram-negative bacterial mutant contained in a secondvial, and instructions for administering the anti-viral agent andattenuated stress-resistant gram-negative bacterial mutant to a subjectfor viral infection prophylaxis or treatment. In accordance with thisembodiment, the attenuated stress-resistant gram-negative bacterialmutant may be engineered to express one or more nucleic acid moleculesencoding one or more therapeutic molecules. Preferably, the attenuatedstress-resistant gram-negative bacterial mutants included in the kits ofthe invention are attenuated stress-resistant gram-negative Salmonellamutants.

In another embodiment, a kit of the invention comprises astress-resistant gram-negative bacterial mutant for viral infectionprophylaxis or treatment in a vial and instructions for administeringthe stress-resistant gram-negative bacterial mutant to a subject, wherethe stress-resistant gram-negative bacterial mutant is a facultativeanaerobe or facultative aerobe. In accordance with this embodiment, thestress-resistant gram-negative bacterial mutant may be engineered toexpress one or more nucleic acid molecules encoding one or moretherapeutic molecules. In another embodiment, a kit of the inventioncomprises an anti-viral agent contained in a first vial, astress-resistant gram-negative bacterial mutant contained in a secondvial, and instructions for administering the anti-viral agent andstress-resistant gram-negative bacterial mutant to a subject with a forviral infection prophylaxis or treatment. In accordance with thisembodiment, the stress-resistant gram-negative bacterial mutant may beengineered to express one or more nucleic acid molecules encoding one ormore therapeutic molecules.

The present invention incorporates a combination of bacterial vector andprotein expression technology which results in a unique vaccine which israpidly constructed in response to emerging avian influenza and theirhighly pathogenic derivatives. The present invention is directed to theconstruction bacterially codon optimized avian and human influenza genesand their incorporation into a Salmonella strain for therapeutic use inthe prevention of avian influenza and highly pathogenic derivatives. Anantigen-expressing plasmid or chromosomal construct in the bacterialstrains described herein may also contain one or more transcriptionalterminators adjacent to the 3′ end of a particular nucleotide sequenceon the plasmid to prevent undesired transcription into another region ofthe plasmid or chromosome. Such transcription terminators thus serve toprevent transcription from extending into and potentially interferingwith other critical plasmid functions, e.g., replication or geneexpression. Examples of transcriptional terminators that may be used inthe antigen-expressing plasmids described herein include, but are notlimited to, the T1 and T2 transcription terminators from 5S ribosomalRNA bacterial genes (see, e.g., FIGS. 1-5; Brosius and Holy, Proc. Natl.Acad. Sci. USA, 81: 6929-6933 (1984); Brosius, Gene, 27(2): 161-172(1984); Orosz et al., Eur. J. Biochem., 20 (3): 653-659 (1991)).

The mutations in an attenuated bacterial host strain may be generated byintegrating a homologous recombination construct into the chromosome orthe endogenous Salmonella virulence plasmid (Donnenberg and Kaper, 1991;Low et al. (Methods in Molecular Medicine, 2003)). In this system, asuicide plasmid is selected for integration into the chromosome by afirst homologous recombination event, followed by a second homologousrecombination event which results in stable integration into thechromosome. The antigen-expressing chromosomal integration constructsdescribed herein comprise one or more nucleotide sequences that encodeone or more polypeptides that, in turn, comprise one or more avianinfluenza antigens, such as the hemagglutinin and neuraminidasepolypeptide antigens, or immunogenic portions thereof, from avianinfluenza virus and highly pathogenic derivatives. Such coding sequencesare operably linked to a promoter of transcription that functions in aSalmonella bacterial strain even when such a bacterial strain isingested, i.e., when a live vaccine composition described herein isadministered orally to an individual. A variety of naturally occurring,recombinant, and semi-synthetic promoters are known to function inenteric bacteria, such as Escherichia coli and serovars of S. enterica(see, e.g., Dunstan et al., Infect. Immun., 67(10): 5133-5141 (1999)).Promoters (P) that are useful in the invention include, but are notlimited to, well known and widely used promoters for gene expressionsuch as the naturally occurring Plac of the lac operon and thesemi-synthetic Ptrc (see, e.g., Amman et al., Gene, 25 (2-3): 167-178(1983)) and Ptac (see, e.g., Aniann et al., Gene, 69(2): 301-315(1988)), as well as PpagC (see, e.g., Hohmann et al., Proc. Natl. Acad.Sci. USA, 92. 2904-2908 (1995)), PpmrH (see, e.g., Gunn et al., Infect.Immun., 68: 6139-6146 (2000)), PpmrD (see, e.g., Roland et al., J.Bacteriol., 176: 3589-3597 (1994)), PompC (see, e.g., Bullifent et al.,Vacccine, 18: 2668-2676 (2000)), PnirB (see, e.g., Chatfield et al.,Biotech. (NY), 10: 888-892 (1992)), PssrA (see, e.g., Lee et al., J.Bacteriol. 182. 771-781 (2000)), PproU (see, e.g., Rajkumari andGowrishankar, J. Bacteriol., 183. 6543-6550 (2001)), Pdps (see, e.g.,Marshall et al., Vaccine, 18: 1298-1306 (2000)), and PssaG (see, e.g.,McKelvie et al., Vaccine, 22: 3243-3255 (2004)), Some promoters areknown to be regulated promoters that require the presence of some kindof activator or inducer molecule in order to transcribe a codingsequence to which they are operably linked. However, some promoters maybe regulated or inducible promoters in E. coli, but function asunregulated promoters in Salmonella. An example of such a promoter isthe well known trc promoter (“Ptrc”, see, e.g., Amman et al., Gene,25(2-3): 167-178 (1983); Pharmacia-Upjohn). As with Plac and Ptac, Ptrcfunctions as an inducible promoter in Escherichia coli (e.g., using theinducer molecule isopropyl-p-D-1 8 thio-galactopyranoside, “IPTG”),however, in Salmonella bacteria having no LacI repressor, Ptrc is anefficient constitutive promoter that readily transcribes avian influenzaantigen-containing polypeptide coding sequences present onantigen-expressing plasmids described herein. Accordingly, such aconstitutive promoter does not depend on the presence of an activator orinducer molecule to express an antigen-containing polypeptide in astrain of Salmonella.

The avian influenza antigen-expressing chromosomal integrationconstructs which integrate into the live vaccine strains also contain anorigin of replication (ori) that enables the precursor plasmids to bemaintained as multiple copies in certain the bacterial cells which carrythe lambda pir element. For the process of cloning DNA, a number ofmulti-copy plasmids that replicate in Salmonella bacteria are known inthe art, as are various origins of replications for maintaining multiplecopies of plasmids. Preferred origins of replications for use in themulti-copy antigen-expressing plasmids described herein include theorigin of replication from the multi-copy plasmid pBR322 (“pBR ori”;see, e.g., Maniatis et al., In Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory, Cold Spring Harbor, 1982), pp. 479-487;Watson, Gene, 70: 399-403, 1988), the low copy origin of replicationfrom pACYC177, and the origin of replication of pUC plasmids (“pUCori”), such as found on plasmid pUC 1 8 (see, e.g., Yanish-Perron etal., Gene, 33: 103-119 (1985)). Owing to the high degree of geneticidentity and homology, any serovar of S. enterica may be used as thebacterial host for a live vaccine composition for avian influenza,provided the necessary attenuating mutations and antigen-expressingplasmids as described herein are also employed. Accordingly, serovars ofS. enterica that may be used in the invention include those selectedfrom the group consisting of Salmonella enterica serovar Typhimurium(“S. typhimurium”), Salmonella montevideo, Salmonella enterica serovarTyphi (“S. typhi”), Salmonella enterica serovar Paratyphi B (“S.paratyphi B”), Salmonella enterica serovar Paratyphi C (“S. paratyphiC”), Salmonella enterica serovar Hadar (“S. hadar”), Salmonella entericaserovar Enteriditis (“S. enteriditis”), Salmonella enterica serovarKentucky (“S. kentucky”), Salmonella enterica serovar Infantis (“S.infantis”), Salmonella enterica serovar Pullorum (“S. pullorum”),Salmonella enterica serovar Gallinarum (“S. gallinarum”), Salmonellaenterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovarAnaturn (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”),Salmonella enterica serovar Derby (“S. derby”), Salmonella entericaserovar Choleraesuis var. kunzendorf (“S. cholerae kunzendorf”), andSalmonella enterica serovar minnesota (“S. minnesota”).

The vaccine compositions described herein may be administered orally toan individual in any form that permits the Salmonella bacterial strainof the composition to remain alive and to persist in the gut for a timesufficient to elicit an immune response to one or more avian influenzaantigens of avian influenza virus and highly pathogenic derivativesexpressed in the Salmonella strain. For example, the live bacterialstrains described herein may be administered in relatively simple bufferor saline solutions at physiologically acceptable pH and ion content. By“physiologically acceptable” is meant whatever is compatible with thenormal functioning physiology of an individual who is to receive a livevaccine composition described herein. Preferably, bacterial strainsdescribed herein are suspended in otherwise sterile solutions ofbicarbonate buffers, phosphate buffered saline (PBS), or physiologicalsaline, that can be easily swallowed by most individuals. However,“oral” routes of administration may include not only swallowing from themouth a liquid suspension or solid form comprising a live bacterialstrain described herein, but also administration of a suspension of abacterial strain through a nasal spray or pulmonary inhaler, anasojejunal or gastrostomy tube, and rectal administration, e.g., byusing a suppository comprising a live bacterial strain described hereinto establish an infection by such bacterial strain in the lowerintestinal tract of the alimentary canal. Accordingly, any of a varietyof alternative modes and means may be employed to administer a vaccinecomposition described herein to the alimentary canal of an individual ifthe individual cannot swallow from the mouth.

7. FIGURES

FIG. 1. Selection scheme for isolation of transposon insertions whichconfer CO₂ resistance. Beginning with the YS1646 strain which is CO₂sensitive, a library of mutants is created using transposon insertionalmutagenesis (e.g., EZ::Tn, Epicentre, Madison, Wis.). The library isthen plated to LB plates and incubated in a 5% CO₂ containingenvironment at 37° C. This results in numerous colonies on the plateswhich are CO₂ resistant, which could be either due to the transposon, ordue to spontaneous mutations. In order to isolate the transposon-relatedCO₂-resistant colonies, the colonies are scraped off the plate usingmedia and a bent glass rod in order to pool the colonies. A phage lysateis prepared from the pooled colonies and used to re-transduce YS1646which is plated to kanamycin. This results in numerouskanamycin-resistant colonies. These colonies are then individuallypatched to a master plate and replica plated to LB and incubated in aCO₂ environment in order to confirm transpon-derived CO₂ resistancephenotype. The retransduction and replica plating is then performed onan individual colony basis. Colonies confirmed to have CO₂ resistanceassociated with the transposon are subjected to genome walkingtechniques which results in identifying the chromosomal insertion site.

FIG. 2. Sensitivity and resistance to CO₂ shown by comparing colonyforming units (CFUs). In each of the three panals, the number ofcolonies on the right is compared with the number of colonies on theleft to indicate sensitivity or resistance. A) Growth of wild typeSalmonella on LB media in either air (left) or CO₂ (right), showing nosensitivity to the CO₂ conditions. B) Growth of VNP20009 (YS1646;41.2.9) on LB media in either air (left) or CO₂ (right) showing strongsensitivity to CO₂. C) VNP20009 zwf on LB media in either air (left) orCO₂ (right) showing that zwf confers resistance to CO₂ of an msbB⁻strain.

FIG. 3. msbB⁻ confers growth sensitivity in liquid media under CO₂conditions containing physiological amounts of salt and is suppressed byzwf⁻. Two sets of Salmonella strains, YS873 and YS873 zwf⁻, and ATCC14028 and ATCC 14028 zwf⁻ were grown on either LB or LB-0 in either airor CO₂. In LB media under ambient air conditions, YS873 and YS873 zwf⁻show a normal growth curve. However, under CO₂ conditions, the YS873strain is highly inhibited and shows as reduction in the number of CFUswhereas the YS873 zwf⁻ strain grows at a much greater rate. B) In LB-0,the CO₂ sensitivity is much less, and is not suppressed by the zwfmuation. C) & D), Wild type Salmonella strain ATCC 14028 and 14028 zwf⁻show similar growth properties in either LB or LB-0 with or without CO₂.

FIG. 4. β-galactosidase release assays confirm cell lysis in LB in thepresence of 5% CO₂ and that zwf confers resistance. Release ofβ-galactosidase from the cytosol of the bacteria was used to test if thedecrease in CFU observed in YS873 in LB in the presence of 5% CO₂resulted from cell lysis. The strains used were Salmonella YS873 andYS873 zwf⁻ grown under either ambient air or 5% CO₂ conditions. After 2hours growth, there is little difference between the strains undereither of the growth conditions. After 6 hours of growth, significantcell lysis, as measured by the release of the cytoplasmic enzymeβ-galactosidase, is observed in YS873 grown in the presence of 5% CO₂.Furthermore, a loss-of-function mutation in zwf significantly reducescell lysis in YS873. No significant cell lysis is observed in theabsence of CO₂.

FIG. 5. zwf suppresses sensitivity to acidic pH in LB broth. Two sets ofSalmonella strains, YS873 and YS873 zwf⁻, and ATCC 14028 and ATCC 14028zwf⁻ were grown on LB at either low pH (pH 6.6) or physiological pH (pH7.6) in either air or 5% CO₂. A) Under ambient air conditions, YS873 isstrongly growth inhibited at pH 6.6, compared to the YS873 zwf⁻ whichsuppresses the inhibition and restores normal growth, while at pH 7.6,both strains grow normally. B) Under 5% CO₂, the zwf mutation suppressedthe sensitivity to acid pH compared to the YS873 strain, which lostviability during the 6 hour time period. Moreover, the zwf mutationchanged the pH optimum of the strain, which now grew better at pH 6.6than at pH 7.6. C) & D), Wild type Salmonella strain ATCC 14028 and14028 zwf⁻ show similar growth properties an either pH 6.6 or pH 7.6with or without CO₂.

FIG. 6. β-galactosidase assays confirm cell lysis in LB broth, pH 6.6and that zwf⁻ confers resistance. Release of β-galactosidase from thecytosol of the bacteria was used to test if the decrease in CFU observedin YS873 in LB at pH 6.6+/− the presence of 5% CO₂ resulted from celllysis. The strains used were Salmonella YS873 and YS873 zwf⁻ grown in LBbroth at either pH 6.5 or pH 7.5 under either ambient air or 5% CO₂conditions. A) Under ambient air conditions after 8 hours, significantcell lysis occurs after growth of YS873 in LB broth, pH 6.5 but not pH7.5. Furthermore, a loss-of-function mutation in zwf significantlyreduces cell lysis of YS873 grown in LB broth pH 6.6. B) Under 5% CO₂conditions after 8 hours, cell lysis is suppressed only in the YS873zwf⁻ strain at pH 6.5, again showing a shift in pH optimum for thisstrain.

FIG. 7. zwf mutation suppresses both msbB-induced CO₂ sensitivity andosmotic defects. Different media and growth conditions were used toindicate the ability of small patches of bacteria (3 each) to grow usingthe replica plating technique. The strains used are listed on the left:wt, wild type Salmonella typhimurium ATCC 14028; YS1, Salmonellatyphimurium ATCC 14028 containing the msbB mutation; YS1 zwf::kan, theYS1 strain with a kanamycin containing transposing insertion into thezwf gene; YS873, the YS1 strain with a deletion in the somA gene; YS873zwf::kan, the YS873 strain with a kanamycin containing Tn5 transposondisrupting the zwf gene. Growth conditions maintained at 37° C. usedincluded: A, LB media in air; B, LB media in 5% CO₂; C, msbB media; D,msbB media in 5% CO₂; E, LB-0 media in air; F, LB-0 media in 5% CO₂; G,LB-0 media containing sucrose (total 455 milliosmoles); H, LB-0 mediacontaining sucrose and 5% CO₂; I, LB-0+gluconate (glucon.) in air; J,LB-0+gluconate in 5% CO₂.

FIG. 8. Selection scheme for isolation of transposon insertions whichconfer acidic pH resistance. Beginning with the YS1646 strain which isacidic pH sensitive, a library of mutants is created using transposoninsertional mutagenesis (e.g., EZ::Tn, Epicentre, Madison, Wis.). Thelibrary is then plated to LB plates at pH≦6.6. This results in numerouscolonies on the plates which are acidic pH resistant, which could beeither due to the transposon, or due to spontaneous mutations. In orderto isolate the transposon-related acidic-resistant colonies, thecolonies are scraped off the plate using media and a bent glass rod inorder to pool the colonies. A phage lysate is prepared from the pooledcolonies and used to re-transduce YS1646 which is plated to kanamycin.This results in numerous kanamycin-resistant colonies. These coloniesare then individually patched to a master plate and replica plated to LBat pH≦6.6 and incubated in order to confirm transposon-derived acidic pHresistance phenotype. The retransduction and replica plating is thenperformed on an individual colony basis. Colonies confirmed to have anacidic pH resistant phenotype associated with the transposon aresubjected to genome walking techniques which results in identifying thechromosomal insertion site.

FIG. 9. Selection scheme for isolation of transposon insertions whichconfer osmolarity resistance. Beginning with the YS1646 strain which isosmolarity sensitive, a library of mutants is created using transposoninsertional mutagenesis (e.g., EZ::Tn, Epicentre, Madison, Wis.). Thelibrary is then plated to LB plates (containing salt). This results innumerous colonies on the plates which are osmolarity resistant, whichcould be either due to the transposon, or due to spontaneous mutations.In order to isolate the transposon-related osmolarity-resistantcolonies, the colonies are scraped off the plate using media and a bentglass rod in order to pool the colonies. A phage lysate is prepared fromthe pooled colonies and used to re-transduce YS1646 which is plated tokanamycin. This results in numerous kanamycin-resistant colonies. Thesecolonies are then individually patched to a master plate and replicaplated to LB and incubated in order to confirm transpon-derivedosmolarity resistance phenotype. The retransduction and replica platingis then performed on an individual colony basis. Colonies confirmed tohave an osmolarity resistant phenotype associated with the transposonare subjected to genome walking techniques which results in identifyingthe chromosomal insertion site.

In order to more fully illustrate the invention, the followingnon-limiting examples are provided.

8. EXAMPLE Isolation and Identification of a Gene Involved in Resistanceto CO₂, Acidic pH and/or Osmolarity 8.1. Example: Isolation of CO₂Resistant Strains Using Transposon Libraries

Throughout the procedures, msbB⁺ strains were grown in Luria-Bertani(LB) broth containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, pHadjusted as indicated using either 1N NaOH or 1N HCl, or LB platescontaining 1.5% agar at 37±2° C. msbB⁻ strains were grown in modified LBreferred to as MSB media (msbB media), containing 10 g tryptone, 5 gyeast extract 2 mL 1N CaCl₂ and 2 mL 1N MgSO₄ per liter, adjusted to pH7.0 to 7.6 using 1N NaOH, or in LB broth or LB plates lacking NaCl,referred to as LB-0. For transductions, LB lacking EGTA was used. Forsucrose resolutions, LB lacking NaCl and containing 5% sucrose at 30±2°C. was used. Auxotrophic mutants are determined on minimal media 56(M56): 0.037 M KH₂PO₄, 0.06 M Na₂HPO₄, 0.02% MgSO₄7H₂O, 0.2% (NH₄)₂SO₄,0.001% Ca (NO₃)₂, 0.00005% FeSO₄7H₂O, with a carbon source (e.g.,glucose 0.1 to 0.3%) as sterile-filtered additive, and furthersupplemented with the appropriate nutrients, 0.1 mg/ml thiamine and 50mg/ml each of adenine. Solid M56 media is made by preparing separateautoclaved 2× concentrates of the mineral salts and the agar, which arecombined after sterilization. Media are also supplemented withantibiotics used as needed to select for resistance markers, includingtetracycline (Sigma) at 4 mg/ml from a stock: 10 mg/ml in 70% ethanolstored in darkness at −20° C. or ampicillin at 100 mg/ml from a stock:100 mg/ml in H₂O, sterile filtered and stored at −20° C. The bacteriaused are listed in Table 1.

TABLE 1 Strain Parental strain Genotype Derivation or source S. entericaATCC 14028 serovar Typhimurium Wild type Wild type Manassas, VA 1402814028 zwf 14028 Δzwf Replacement of zwf gene with Δzwf by homologousrecombination YS1646 14028 ΔmsbB ΔpurI Low et al., pp 47-59, In: SuicideGene (VNP20009) Therapy: Methods and Protocols, C. Springer (ed), HumanaPress, 2003. YS1 14028 msbB1::Ωtet Murray et al. 2001, J. Bacteriol.183: 5554-5561. YS1 zwf YS1 msbB1::Ωtet P22 zwf:Tn5 (Kan^(R))X YS1 →Kan₂₀ ^(r) zwf:Tn5 (Kan^(R)) YS873 14028 msbB1::Ωtet somA Murray et al.2001, J. Bacteriol. 183: zbj10:Tn10 5554-5561. YS873 zwf YS873msbB1::Ωtet somA P22 zwf:Tn5 (Kan^(R))X YS873 → Kan₂₀ ^(r) zbj10:Tn10zwf:Tn5 (Kan^(R))

CO₂ resistant mutants were obtained as outlined in FIG. 1. A Tn5transposome (EZ::TN, Epicentre, Madison, Wis.) was used to directlygenerate a library in YS1646, plated to MSB agar plates with theappropriate antibiotic (kanamycin for Tn5) and grown overnight at 37° C.in ambient air. The plates were then flooded with MSB broth and thecolonies scraped from the plates, pooled and frozen in aliquots at −80°in 15% glycerol.

The library was screened by plating dilutions of the library onto MSBagar and incubating them in 5% CO₂ at 37° C. overnight. In particular,colonies were tested for CO₂ resistance by plating serial dilutions ofthe library of bacteria onto MSB plates and incubating the plates at 37°C. in either ambient air or in air with 5% CO₂. Colonies that wererecovered from MSB plates incubated at 37° C. in air with 5% CO₂ weredeemed resistant to CO₂. The resistance of these colonies to CO₂ couldbe due to either the presence of the transposon insertion or to aspontaneous mutants. In order to eliminate any background of spontaneousmutants, the CO₂-resistant colonies were pooled, P22 lysates prepared(P22 phage transduction by the method of Davis et al., 1980, AdvancedBacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.),and the Tn5 insertions transferred to YS1646 and plates for individualcolonies in MSB-kanamycin at 37° C. in ambient air. Individuals colonieswere then gridded and replica plated to MSB plates and incubated at 37°C. in either ambient air or in air with 5% CO₂. Those colonies whichtested positive by the replica plating were chosen for further study ofretransduction to confirm the phenotype. These clones were also testedto ensure that there was no significant increase in TNF-α inductionusing standard techniques such as those described by Low et al., 1999.The CO₂ resistant clones chosen to undergo further testing include theclones designated 14.2, 32.2 and 37.2.

An example of the CO₂ sensitivity and resistance observed using theplating efficiency method is shown in FIG. 2. The percent growth of thebacteria under a stress condition such as CO₂ is determined by platingto MSB agar plates and incubated in either air or CO₂, and dividing thenumber of clones on the stress-subjected plate to the number of clonesin the non-stress-subjected plate. General observation of the plate isoften sufficient to determine sensitivity and resistance. The wild typebacteria do not show any obvious reduction in the number of CFUsobserved, whereas the strain YS1646 shows a dramatic reduction in thenumber of bacteria observed. The CO₂ resistant mutant 32.2 showsapproximately the same number of colonies when grown either underambient air conditions or 5% CO₂, indicating CO₂ resistance. TheGenomeWalker (Clonetech, Palo Alto, Calif.) kit was used to determinethe chromosomal insertion site in clones designated 14.2, 32.2 and 37.2.The Tn5 was determined to be located in the zwf gene in all threeclones, two of them (clones 14.2 and 32.2) were located after base pair1019 and the third (clone 37.2) was located after base pair 1349.Therefore, zwf⁻ confers CO₂ resistance in the msbB⁻ strain YS1646.

8.2. Example: Suppression of CO₂-Mediated Growth Inhibition by zwf⁻Mutants

In order to further analyze the effect of the zwf mutation on growth ofthe msbB⁻ Salmonella, growth rates under different conditions werestudied. To generate growth curves, 3 ml broth tubes were inoculatedwith single colonies and grown on a shaker overnight at 37° C. Anadequate amount of LB or LB-0 broth was then inoculated 1:1000 withcells. Cells were held on ice until all inoculations were completed.Triplicate 3 ml aliquots were then placed in a 37° C. shaker with 250rpm in air or 5% CO₂. O.D.₆₀₀ was measured every 60 minutes anddilutions of bacteria were plated onto MSB or LB agar plates tocalculate the number of colony forming units (CFU) per ml.

FIG. 3 shows the growth of wild type ATCC 14028, 14028 zwf⁻, YS873, andYS873 zwf⁻ in LB and LB-0 broth, grown in the presence or absence of 5%CO₂. The growth of YS873 (FIG. 3A), but not ATCC 14028 (FIG. 3C) isgreatly impaired LB broth in the presence of 5% CO₂. A significantdecrease in CFU is observed (FIG. 3A), indicating that YS873 cells loseviability in the presence of 5% CO₂ in LB broth. When a loss-of-functionmutation in zwf is incorporated into YS873, no loss in viability isobserved under identical conditions, although there is a longer lagphase of growth and the CFU does not increase at the same rate as in LBbroth in the absence of 5% CO₂ (FIG. 3A). In LB-0 broth, there are nogrowth defects in 14028 or 14028 zwf⁻ (FIG. 3D). For YS873 and YS873zwf⁻, the growth defects in LB-0 in the presence of 5% CO₂ areattenuated in comparison to those observed in LB broth. There is nodecrease in viability in YS873 in LB-0 in 5% CO₂, although there is adecreased growth rate in both YS873 and YS873 zwf⁻ in LB-0 in thepresence of CO₂ compared to growth in the absence of CO₂ (FIG. 3B).

8.3. Example: Suppression of CO₂ Mediated Cell Lysis by zwf⁻ Mutants

To test if the decrease in CFU observed in YS873 in LB in the presenceof 5% CO₂ resulted from cell lysis, release of β-galactosidase (acytoplasmic enzyme not normally present in the culture supernatant) wasdetermined. For β-galactosidase expression, lacZ was cloned into thehigh copy vector pSP72 (Promega) and screened for bright blue colonieson LB agar containing 40 μg/ml X-gal. β-gal assays were performedaccording to the instructions for the Galacto-Star™ chemiluminescentreporter gene assay system (Applied Biosystems, Bedford, Mass.).Briefly, 1 ml of bacterial culture expressing β-Gal from pSP72 waspelleted at 13,000×g for 5 min. Supernatants were filtered through a 0.2mm syringe filter and then assayed immediately or frozen at −80° C.until assayed with no further processing. Cell pellets were quicklyfreeze-thawed and suspended in 50 ul or 200 ml B-Per™ bacterial celllysis reagent (Pierce Chemical) containing 10 mg/ml lysozyme (Sigma).Bacteria were allowed to lyse for 10-20 min. at room temperature andthen placed on ice. All reagents and samples were allowed to come toroom temperature before use. Filtered supernatants and bacterial lysateswere diluted as needed in Galacto-Star™ Lysis Solution or assayeddirectly. β-gal standard curves were made by preparing recombinant β-gal(Sigma, 600 units/mg) to 4.3 mg/ml stock concentration in 1×PBS. Thestock was diluted in Lysis Solution to prepare a standard curve of 100ng/ml-0.05 ng/ml in doubling dilutions. 20 ml of standard or sample wasadded to each well of a 96-well tissue culture plate. 100 ml ofGalacto-Star™ Substrate diluted 1:50 in Reaction Buffer Diluent wasadded to each well and the plate rotated gently to mix. The plate wasincubated for 90 minutes at 25° C. in the dark and then read for 1second/well in an L-max™ plate luminometer (Molecular Devices). Samplelight units/ml were compared to the standard curve and values convertedto units β-gal/ml. Percent release of β-gal was determined by dividingunits/ml supernatant by total units/ml (units/ml supernatant+units/mlpellet). All samples were assayed in triplicate. As shown in FIG. 4,after 6 hours of growth, significant cell lysis, as measured by therelease of the cytoplasmic enzyme β-galactosidase, is observed in YS873grown in the presence of 5% CO₂. No significant cell lysis is observedin the absence of CO₂. Furthermore, a loss-of-function mutation in zwfsignificantly reduces CO₂-mediated cell lysis in YS873.

8.4. Example: Suppression of Acidic pH Mediated Growth Inhibition by zwfMutants.

To test if increased or reduced pH would reduce sensitivity to CO₂, LBmedia was buffered to pH 6.6 or 7.6 and cultures were grown in thepresence or absence of 5% CO₂. As shown in FIG. 5, wild type ATCC 14028and ATCC 14028 zwf⁻ grow normally under all conditions (FIGS. 5C & 5D).In contrast, the growth of YS873 is significantly impaired when the pHof LB is 6.6 under ambient air conditions, with no significant increasein CFU after 6 hours (FIG. 5A). In contrast, when the pH of LB is 7.6,YS873 grows well (FIG. 5A). A loss-of-function mutation in zwf allowsfor YS873 to grow well in LB broth at a pH of 6.6 (FIG. 5A). Under 5%CO₂, the zwf mutation suppressed the sensitivity to acid pH compared tothe YS873 strain (FIG. 5B), which lost viability during the 6 hour timeperiod. Moreover, the zwf mutation changed the pH optimum of the strain,which now grew better at pH 6.6 than at pH 7.6.

8.5. Example: Suppression of Acidic pH Mediated Cell Lysis by zwfMutants

Release of β-galactosidase from the cytosol of the bacteria was used totest if the decrease in CFU observed in YS873 in LB at pH 6.6+/− thepresence of 5% CO₂ resulted from cell lysis (FIG. 6). The strains usedwere Salmonella YS873 and YS873 zwf⁻ grown in LB broth at either pH 6.5or pH 7.5 under either ambient air or 5% CO₂ conditions. Under ambientair conditions after 8 hours, significant cell lysis occurs after growthof YS873 in LB broth, pH 6.5 but not pH 7.5 (FIG. 6A). Furthermore, aloss-of-function mutation in zwf significantly reduced cell lysis ofYS873 grown in LB broth pH 6.6. Under 5% CO₂ conditions after 8 hours,cell lysis is suppressed only in the YS873 zwf⁻ strain at pH 6.5, againshowing a shift in pH optimum for this strain (FIG. 6B).

8.6. Example: Determination of Osmolarity and Gluconate SensitivityProperties of zwf Mutants

Phenotypes of strains were determined by replica plating (FIG. 7).Master plates were made on either MSB or LB-0 agar. Plates weresupplemented with sucrose (455 mOsmol) instead of NaCl, or 0.33%gluconate, a downstream product in the same pathway as zwf, thusrestoring or enhancing the pathway using a metabolic supplement. Replicaplating was performed using a double velvet technique. Plates wereincubated for 16 hours at 37° C. in either ambient air or 5% CO₂. Forcomparative purposes, the wild type Salmonella ATCC 14028, YS1, YS1zwf::kan, YS873 and YS873 zwf::kan were used.

As shown in the replica series of FIG. 7, growth of unsuppressed YS1 isinhibited on LB (FIG. 7A) but YS1 grew well on MSB and LB-0 agar (FIGS.3C and 3D. In contrast, growth of YS1 on MSB and LB-0 agar is completelyinhibited when the plates are incubated in the presence of 5% CO₂. Theintroduction of the zwf mutation completely compensates for thephenotype and allows the bacteria to grow under 5% CO₂ on all threemedia (FIG. 7 B, D, F). When NaCl in LB plates is substituted withsucrose at iso-osmotic concentrations (FIG. 7 G), growth of YS1 is alsoinhibited, indicating osmosensitivity of YS1. Introduction of the zwfmutation improves growth of YS1 on LB and on LB-0 5% sucrose agar,indicating that the zwf mutation can partially compensate for themsbB-induced osmotic growth defect. YS873, which contains the EGTA andsalt resistance suppressor mutation somA (Murray et al., 2001), growswell on LB, MSB, LB-0 and LB-0 sucrose agar plates in air, but not whenthe plates are incubated in 5% CO₂. In contrast, the strain YS873 zwf⁻is able to grow on all plates in CO₂, indicating that the zwf mutationcan compensate for the growth defect of msbB strains in CO₂. YS873 zwf⁻was not able to grow on LB-0 gluconate in 5% CO₂ (FIG. 7 I +J),confirming the role of the zwf pathway in CO₂ sensitivity.

8.7. Example: Isolation and Identification of Genes Involved inResistance to Acid pH

The Tn5 insertion library described above is screened to identifymutants with resistance to acidity FIG. 8. The library is screened bydilution of the library onto MSB agar or broth buffered to pH 6.6 with100 mM sodium phosphate buffer and incubating them at 37° C. overnight.Colonies that were recovered from acidified MSB plates incubated at 37°C. in air are deemed acidic pH resistant. The resistance of thesecolonies could be due to either the presence of the transposon insertionor to spontaneous mutants. In order to eliminate any background ofspontaneous mutants, the acidic pH-resistant colonies are pooled, P22lysates prepared (P22 phage transduction by the method of Davis et al.,1980, Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.), and the Tn5 insertions transferred to YS1646 and platedfor individual colonies in pH 7.4 MSB-kanamycin at 37° C. in ambientair. Individuals colonies were then gridded and replica plated to pH 6.6and pH 7.4 MSB plates and incubated at 37° C. Those colonies whichtested positive by the replica plating are chosen for further study ofretransduction to confirm the phenotype. These clones are also tested toensure that there was no significant increase in TNF-α induction usingstandard techniques such as those described by Low et al., 1999. TheGenomeWalker (Clonetech, Palo Alto, Calif.) kit is used to determine thechromosomal insertion site in the acidic pH-resistant clones.

8.8. Example: Isolation and Identification of Genes Involved inResistance to Osmolarity

The Tn5 insertion library described above is screened to identifymutants with resistance to osmolarity FIG. 9. The library is screened bydilution of the library onto MSB agar or broth containing sucrose suchthat it results in greater than 100 mOsmoles, at physiologicalosmolarity, (approx 300 mOsmole) or greater (e.g., 450 mOsmole) andincubating them at 37° C. overnight. Colonies that were recovered fromphysiological osmolarity or greater on MSB plates incubated at 37° C. inair are deemed osmolarity resistant. The resistance of these coloniescould be due to either the presence of the transposon insertion or to aspontaneous mutants. In order to eliminate any background of spontaneousmutants, the osmolarity-resistant colonies are pooled, P22 lysatesprepared (P22 phage transduction by the method of Davis et al., 1980,Advanced Bacterial Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y.), and the Tn5 insertions transferred to YS1646 and platedfor individual colonies in MSB-kanamycin at 37° C. in ambient air.Individuals colonies were then gridded and replica plated to MSB andMSB-sucrose plates and incubated at 37° C. Those colonies which testedpositive by the replica plating are chosen for further study ofretransduction to confirm the phenotype. These clones are also tested toensure that there was no significant increase in TNF-α induction usingstandard techniques such as those described by Low et al., 1999. TheGenomeWalker (Clonetech, Palo Alto, Calif.) kit is used to determine thechromosomal insertion site in the osmolarity-resistant clones.

9. EXAMPLES OF INCORPORATION OF STRESS RESISTANCE GENES FOR USE INGENETICALLY STABILIZED AND ISOLATED STRAINS WITH DEFINED FLAGELLARANTIGENS AND THEIR USE IN PROTECTION AGAINST AVAIN INFLUENZA AND HIGHLYPATHOGENIC DERIVATIVES 9.1. Example. Construction of anAntibiotic-Sensitive Non-Polar Deletion in zwf

A non-polar deletion in zwf was generated by constructing a pCVD442vector capable of deleting the entire zwf coding region by homologousrecombination with the Salmonella chromosome (Donnenberg and Kaper, 1991Infection & Immunity 59:4310-4317; Low et al., 2003, Methods inMolecular Medicine; Suicide Gene Therapy, C. Springer (ed), HumanaPress, pp 47-59, expressly incorporated in their entirety herein).Primers for PCR were designed that would generate one productimmediately upstream of the 5′ ATG start codon and a separate productimmediately downstream of the 3′ stop codon of the zwf coding region.The two separate products could then be ligated sequentially into thepCVD442 vector. The primers were: zwf-5′-reverse: 5′-GTG TGA GCT CGT GGCTTC GCG CGC CAG CGG CGT TCC AGC-3′ (SEQ ID No.:1) (with added Sad) andzwf-5′ forward 5′-GTG TGC ATG CGG GGG GCC ATA TAG GCC GGG GAT TTA AATGTC ATT CTC CTT AGT TAA TCT CCT GG-3′ (SEQ ID No.:2) (with added SphI);and zwf-3′ reverse 5′-GTG TGC ATG CGG GGT TAA TTA AGG GGG CGG CCG CATTTG CCA CTC ACT CTT AGG TGG-3′ (SEQ ID No.:3) and 3-forward 5′-GTG TGTCGA CCC TCG CGC AGC GGC GCA TCC GGA TGC-3′ (SEQ ID No.:4). The primersalso generate internal Not1, Pad, SphI, SfiI, and SwaI in order tofacilitate cloning of DNA fragments, such as the influenza H5 and N1antigens into the Δzwf for stable chromosomal integration withoutantibiotic resistance. This vector is referred to as pCVD442-Δzwf.Presence of the deletion, in Amp^(S) Suc^(R) colonies, was detected withPCR using the following primers: zwf-FL-forward:5′-ATATTACTCCTGGCGACTGC-3′ (SEQ ID No.:5) and zwf-FL-reverse:5′-CGACAATACGCTGTGTTACG-3′ (SEQ ID No.:6).

9.2. Example: Determination of Improved Penetration and Persistence inGut Tissues

Standard methods are utilized to determine increased penetration andpersistence in gut tissues. In all cases, comparison of different doselevels are performed, comparing the parental, CO₂, acidic pH, and/orosmolarity sensitive strain with the CO₂, acidic pH and/or osmolarityresistant strain(s). 1) Total recovery from gut material. Mice areorally administered the parental strain and the resistant strains atdifferent dose levels. At fixed times between days 1 and 21 (e.g., d. 1,7, 14 & 21) the mice are euthanized (avoiding CO₂ asphyxiation) andtheir gut collected by dissection. The gut is then homogenized andserial dilutions plated for Salmonella on Salmonella selective mediasuch as SS agar, bismuth sulfite agar, or Hecktoen enteric agar. Thenumber of Salmonella present for the parental and resistant strains atdifferent times and dosing levels can then be compared to demonstrateimproved penetration and persistence in the gut. 2) Determination of gutlining-associated Salmonella. Mice are orally administered the parentalstrain and the resistant strains at different dose levels. At fixedtimes between days 1 and 21 (e.g., d. 1, 7, 14 & 21) the mice areeuthanized (avoiding CO₂ asphyxiation) and their gut collected bydissection. The gut is then repeated flushed with a saline solutioncontaining 100 ug/ml of gentamicin, an antibiotic that does not entercells and will therefore not kill any bacteria that have penetrated thegut mucosal cells. The gut is then washed with saline to remove tracesof gentamicin, homogenized and serial dilutions plated for Salmonella onSalmonella selective media such as SS agar, bismuth sulfite agar, orHecktoen enteric agar. The number of Salmonella present for the parentaland resistant strains at different times and dosing levels can then becompared in order to demonstrate improved gut penetration andpersistence in the gut at lower doses.

9.3. Example: Determining Immune Response to H5N1-Expressing Bacteria

Live bacterial vaccines for H5N1 influenza prophylaxis or treatmentdescribed by Bermudes (WO/2008/03908) are engineered as described aboveto have an additional mutation in a stress-resistance gene such as zwf.Experimental determination of vaccine activity is known to those skilledin the arts. By way of non-limiting example, determination of anantibody response is demonstrated.

1) Vertebrate animals including mice, birds, dogs, cats, horses, pigs orhumans are selected for not having any known current or recent (within 1year) influenza infection or vaccination. Said animals are pre-bled todetermine background binding to, for example, H5 and N1 antigens.

2) The Salmonella expressing H5 and N1 are cultured on LB agar overnightat 37°. Bacteria expressing other H and or N antigens may also be used.

3) The following day the bacteria are transferred to LB broth, adjustedin concentration to OD₆₀₀=0.1 (˜2×10⁸ cfu/ml), and subjected to furthergrowth at 37° on a rotator to OD₆₀₀=2.0, and placed on ice, where theconcentration corresponds to approx. 4×10⁹ cfu/ml.

4) Following growth, centrifuged and resuspended in 1/10 the originalvolume in a pharmacologically suitable buffer such as PBS and they arediluted to a concentration of 10⁴ to 10⁹ c.f.u./ml in apharmacologically suitable buffer on ice, warmed to room temperature andadministered orally or intranasally in a volume appropriate for the sizeof the animal in question, for example 50 μl for a mouse or 10 to 100 mlfor a human. The actual dose measured in total cfu is determined by thesafe dose as described elsewhere in this application.

5) After 2 weeks, a blood sample is taken for comparison to thepretreatment sample. A booster dose may be given. The booster may be thesame as the initial administration, a different species, a differentserotype, or a different flagellar antigen (H1 or H2) or no flagellarantigen.

6) After an additional 2 to 4 weeks, an additional blood sample may betaken for further comparison with the pretreatment and 2 week posttreatment.

7) A comparison of preimmune and post immune antibody response ispreformed by immunoblot or ELISA. A positive response is indicated by arelative numerical value 2× greater then background/preimmune assay.

9.4. Example: Immunization with H5N1 Bacterial Vaccine Strains

Live bacterial vaccines for H5N1 influenza prophylaxis or treatmentdescribed by Bermudes (WO/2008/03908) are engineered as described aboveto have an additional mutation in a stress-resistance gene such as zwf.An experiment to determine if H5N1 strains of Salmonella are capable ofproviding protection from challenge with the wildtype strain. Ducks areimmunized orally with 5×10⁹ cfu of bacteria when 4 weeks old, thenchallenged with the standard challenge model of avian influenza at 6weeks age.

Birds in Group A are immunized with empty vector. Group B receiveSalmonella H5N1. Group C is immunized with Salmonella expressing theTamiflu resistant neuraminidase mutations. Birds in Group D are notimmunized. Each group is further divided into +/−Tamiflu treatment.Results of these experiments can be used to demonstrate theeffectiveness of the vaccine on Tamiflu resistant strain, with andwithout Tamiflu treatment.

9.5. Example: Immunization with a trimeric hemagglutinin antigen

The bacteria described in Example 9.4 “Immunization with H5N1 BacterialVaccine Strains” above, are further engineered to contain a trimericimmunogen described by Wei et al. 2008 (J Virology 82: 6200-6208,expressly incorporated by reference in its entirety herein). The antigenis further modified to contain the HlyA C-terminal 60 amino acidsin-frame, in order to guide secretion together with HlyBD (and afunctional tolC). Immunization and efficacy evaluations are performed asdescribed above.

9.6. Example: Control of Bacterial Infection with Gluconate

As described herein, in an msbB⁻ zwf⁻ strains are sensitive tophysiological concentrations of CO₂ in the presence of gluconate. Theability of gluconate to control excessive bacterial infections, such asmight occur in a patient who becomes immunocompromised or otherwise hastheir health complicated such that the proliferation of the bacteriarequires control, can be modeled using immunocompromised mice, such asnude (nu/nu) or severe combined immunodeficient (SCID) mice.

1) The msbB⁻ zwf⁻ Salmonella cultured on LB agar overnight at 37°.

2) The following day the bacteria are transferred to LB broth, adjustedin concentration to OD₆₀₀=0.1 (˜2×10⁸ cfu/ml), and subjected to furthergrowth at 37° on a rotator to OD₆₀₀=2.0, and placed on ice, where theconcentration corresponds to approx. 4×10⁹ cfu/ml.

3) Following growth, centrifuged and resuspended in 1/10 the originalvolume in a pharmacologically suitable buffer such as PBS and they arediluted to a concentration of 10⁴ to 10⁹ c.f.u./ml in apharmacologically suitable buffer on ice, warmed to room temperature andadministered orally or intranasally in a volume appropriate for the sizeof the animal in question, for example, not more than 10 μl/g bodyweight for a mouse. The actual dose measured in total cfu is determinedby the safe dose as described by Bermudes (WO/2008/03908), dependingupon the stain of bacteria.

4) A dose response of 1, 5, 50 or 100 mg/mouse (50 mg, 250 mg, 2.5 g, 5g/kg) of gluconate is given either orally or by intravenousadministration.

5) After 1 week, a blood or tissue sample is taken for comparison to thepretreatment sample using a colony forming unit assay.

6) Survival of the mice is monitored over time.

Other Embodiments

Other embodiments are within the claims set forth below. For example,the host bacterium can be E. coli or any other lipid mutant or non-lipidmutant enteric bacterium found to be sensitive to CO₂, acidic pH and/orosmolarity, including those of the genera Salmonella, Bordetella,Shigella, Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella,Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas, all of whichare known or believed to have cell wall structures similar to E. coliand Salmonella.

The various aspects of the disclosure may be combined and subcombined torepresent all consistent combinations and subcombinations withoutdeparting from the scope of the invention. The invention is limited byneither the specific embodiments of the specification, nor theparticular scope of the claims, but rather is to be treated asencompassing the full scope of each aspect disclosed, and the variouscombinations and permutations, which do not depart from the enableddisclosure herein.

1. A mutant Salmonella sp. comprising a genetically modified msbB geneand a mutation characterized by an increased growth when grown under atleast one of high CO₂ conditions, low pH media, and high osmolarity,compared to the msbB- mutant Salmonella designated YS1646.
 2. The mutantSalmonella sp. according to claim 1, further comprising a geneticallymodified zwf gene.
 3. The mutant Salmonella sp. according to claim 2,capable of being inhibited or killed by the addition of gluconate at alevel which does not kill or inhibit wild type Salmonella.
 4. The mutantSalmonella sp. for viral prophylaxis or treatment of claim 1 whichfurther comprises one or more genetically modified genes to auxotrophy.5. The mutant Salmonella sp. for viral prophylaxis or treatment of claim4, wherein at least one of the genetically modified genes to auxotrophyis a gene in a biosynthetic pathway selected from the group consistingof the isoleucine biosynthetic pathway, valine biosynthetic pathway,phenylalanine biosynthetic pathway, tryptophan biosynthetic pathway,tyrosine biosynthetic pathway, and arginine biosynthetic pathway.
 6. Themutant Salmonella sp. for viral prophylaxis or treatment of claim 4,wherein at least one of the genetically modified genes to auxotrophy isa genetically modified AroA gene.
 7. The mutant Salmonella sp. for viralprophylaxis or treatment of claim 4, wherein at least one of thegenetically modified genes to auxotrophy is a gene in a uracil or purinebiosynthesis pathway.
 8. The mutant Salmonella sp. for viral prophylaxisor treatment of claim 1 of which is engineered to contain one or morenucleic acid molecules encoding one or more therapeutic agents.
 9. Apharmaceutical composition comprising an amount of the mutant Salmonellasp. of claim 1 effective to prevent or treat a virally induced diseaseand a pharmaceutically acceptable carrier.
 10. A method for viralprophylaxis or treatment in a subject, said method comprisingadministering to a subject in need thereof an effective amount of themutant Salmonella sp. of claim
 1. 11. A method for limiting the growthof a bacterial strain comprising the addition of gluconate to a mediafor the bacterial strain.
 12. A live Salmonella bacteria, that isgenetically engineered to express one or more viral antigens capable ofviral prophylaxis or treatment having a first attenuating mutation thatreduces TNF-α induction but confers sensitivity to CO₂, osmolarityand/or acidic pH and a second mutation that confers resistance to CO₂,osmolarity and/or acidic pH that maintains reduced TNF-α induction. 13.The live Salmonella bacterium according to claim 12, having improvedability to establish a population (infection) in the gut and, ifproperly modified they could provide a desirable source of immunogenicavian influenza antigen polypeptide(s) to elicit an immune response inthe mucosal tissue of the individual.